From the Departments of Biochemistry and § Medicine/Infectious Disease, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157-1016
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ABSTRACT |
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Acetyl-CoA:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine
acetyltransferase, along with phospholipase A2, is a key
regulator of platelet-activating factor biosynthesis via the remodeling pathway. We have now obtained evidence in human neutrophils indicating that this enzyme is regulated by a specific member of the
mitogen-activated protein kinases, namely the p38 kinase. We earlier
demonstrated that tumor necrosis factor- The platelet-activating factor
(PAF),1
1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine,
is a potent lipid mediator eliciting responses in a wide variety of
cell types (1-3). It is a major mediator of inflammation often acting
in coordination with arachidonic acid metabolites (4-9). Much work has
been devoted to determine how inflammatory lipid mediators are formed
and their production regulated. In human neutrophils and other
inflammatory cells, the PAF is synthesized in response to a variety of
stimuli by a remodeling pathway in which
1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (1-alkyl-2-AA-GPC) is converted to lyso-PAF (1-alkyl-2-lyso-GPC), which
is then acetylated by acetyl-CoA:lyso-PAF acetyltransferase to form
PAF. Physiologically, it is not clear whether the lyso-PAF is primarily
generated by the direct action of PLA2 on 1-alkyl-2-AA-GPC (1, 10) or whether PLA2 first hydrolyzes AA from the
ethanolamine plasmalogen, initiating a CoA-independent
transacylase-catalyzed reaction that transfers AA from 1-alkyl-2-AA-GPC
to the lyso-plasmenylethanolamine generating lyso-PAF by an indirect
route (11). In either the direct or indirect routes, the actions of
both PLA2 and the acetyltransferase are required for PAF synthesis.
Acetyl-CoA:lyso-PAF acetyltransferase was initially demonstrated and
partially characterized by Wykle et al. (12). The enzyme was
found to be present in several rat tissues with the highest activity
found in spleen. Furthermore, the reaction could be inhibited by
divalent cations (Ca2+, Mn2+, and
Mg2+). Both 1-O-alkyl- and
1-O-acyl-2-lyso-sn-glycero-3-phosphocholine could
act as substrates, but
3-O-alkyl-2-lyso-sn-glycero-1-phosphocholine could not, indicating the stereospecificity of this enzyme. It was
later shown that this activity could be increased in PMN stimulated by
A23187 (13), opsinized zymosan (13, 14), and PAF itself (15). Increases
in acetyltransferase activity have also been observed in eosinophils
from patients with eosinophilia compared with cells isolated from those
with normal eosinophil numbers (16). Subsequent to these findings, it
was proposed by Lenihan and Lee (17) that the activation of
acetyltransferase is regulated in a reversible activation/inactivation
manner by phosphorylation. They showed that rat spleen microsomal
acetyltransferase activity could be enhanced by the addition of the
soluble fraction from rat spleen in the presence of ATP and
Mg2+ (17). Moreover, the addition of phosphatidylserine,
diolein, and Ca2+ further enhanced the activity of
acetyltransferase, leading the authors to speculate that protein kinase
C may be involved in regulation of the acetyltransferase.
Gomez-Cambronero et al. (18) later showed that rat spleen
microsomal acetyltransferase activity could be increased by the
catalytic subunit of cyclic AMP-dependent protein kinase in
the presence of Mg2+ and ATP; subsequent treatment of the
microsomal preparations with alkaline phosphatase abolished the
observed increase. It has also been shown that
calcium/calmodulin-dependent protein kinases can lead to
increased activation of acetyltransferase in vitro (19).
These earlier findings provided evidence that acetyltransferase
activity is modulated by phosphorylation but have led to an unclear
picture as to the specific kinase responsible for activation of the
acetyltransferase in vivo. We now have obtained evidence in
human neutrophils that a member of the MAP kinase family, namely p38
kinase, is responsible for the phosphorylation, and hence, activation
of acetyltransferase. The recent development of specific inhibitors of
p38 kinase (20), as well as the availability of purified recombinant,
activated p38 enzyme, has allowed us to demonstrate that this enzyme
activates acetyltransferase in whole cells and in a cell-free system.
Materials--
1-O-[1,2-3H]Hexadecyl-2-lyso-GPC
(56 Ci/mmol) was synthesized as described previously (21), except that
16:0 plasmenylcholine isolated from beef heart by reverse phase high
performance liquid chromatography was used instead of
1-O-hexadec-9'-enyl-2-lyso-GPC. Unlabeled 1-alkyl-lyso-GPC
and other phospholipid standards were purchased from Avanti Polar
Lipids, Inc. (Birmingham, AL). Dextran T-500 was from Amersham
Pharmacia Biotech (Uppsala, Sweden). Isolymph was obtained from
Gallard-Schlesinger (Carle Place, NY). Silica Gel G plates were from
Analtech. PD 98059 and SB 203580 were obtained from
Calbiochem-Novabiochem Intl. Recombinant, constitutively activated p38,
ERK-1, and ERK-2 were obtained from Upstate Biotechnology Inc.(Lake
Placid, NY). Tumor necrosis factor- Preparation of Neutrophils--
Neutrophils were prepared from
heparinized, venous blood collected from healthy, medication-free
donors using dextran sedimentation, isolymph sedimentation, and brief
hypotonic lysis to remove red blood cells as described previously (22).
The resulting cell population consisted of >95% PMN.
Acetyltransferase Activity Measurements--
PMN (1 × 107/ml, 3 ml total) in phosphate-buffered saline containing
1.4 mM CaCl2 was incubated for 30 min at
4 °C, warmed at 37 °C for 5 min, treated with or without SB
203580 and/or PD 98059 for 30 min at 37 °C, and stimulated with the
various agonists as detailed in figure legends. Reactions were
terminated by the addition of 30 ml of ice-cold phosphate-buffered
saline without CaCl2, and cells were pelleted at 300 × g for 10 min at 4 °C. Pelleted cells were resuspended
in 1 ml of protection buffer (0.2 M Tris-HCl (pH 7.5),
containing 50 µg/ml leupeptin, 50 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF,
0.2 mM Na3VO4) and sonicated twice
for 8 s at a power setting of 2 and 10% output with a probe sonicator
(Heat System Inc.).
Acetyltransferase activity was determined according to the method of
Wykle et al. (12). Freshly isolated whole cell sonicates (100 µl, ~75-100 µg of total protein) were incubated with
acetyl-CoA (100 µM) and [3H]lyso-PAF (16 µM, 0.1 µCi/tube in 1 ml final volume). Reactions were
carried out at 37 °C for 15 min and terminated by extraction of the
lipids (23). [3H]PAF was measured by chromatography
Extraction and Chromatography of Lipids--
Lipids were
extracted by a modified Bligh and Dyer procedure (23) in which 9%
formic acid was added to lower the pH to between 3 and 4. Lipids were
dried under a stream of nitrogen, resuspended in chloroform:methanol
(4:1, v/v), and separated on Silica Gel G layers developed in
chloroform:methanol:acetic acid:water (50:25:8:4, v/v/v/v).
Phospholipids and standards were visualized by exposure to iodine
vapor, whereas radiolabeled products were detected using a
radiochromatogram imaging system (Bio-scan Inc., Washington, D.C.). For
quantitation of radioactivity, individual lipid classes were scraped
from thin layer plates and counted by liquid scintillation counting
(Minaxi Tri-Carb 4000 series).
In Vitro Kinase Activation of Acetyltransferase--
PMN
(7.5 × 107/ml) were treated with or without 1 nM TNF-
Isolated membranes (40 µl, ~5 µg of total protein) from untreated
cells were immediately incubated at 37 °C with 2 µg of
recombinant, activated p38, ERK-1, or ERK-2 in the presence of 500 µM Mg2+ and 40 µM ATP in a
final volume of 50 µl. After 15 min, the entire 50 µl of kinase
reaction mixture was added to a vessel containing acetyl-CoA (100 µM) and [3H]lyso-PAF (16 µM,
0.1 µCi/tube) in 950 µl of 0.2 M Tris-HCl (pH 7.5) and
incubated at 37 °C. The reaction mixtures were extracted after 15 min and analyzed for [3H]PAF formation as described
above. As a positive control, microsomes from TNF- Alkaline Phosphatase Treatment of PMN Membranes--
Isolated
membranes (15 µl, ~5 µg of total protein) from either control
cells or cells treated with TNF- Activation of MAP Kinase(s) and Acetyltransferase by TNF-
We have now extended our studies to focus on the activation of
acetyl-CoA:lyso-PAF acetyltransferase using the three test stimuli
previously examined. Human PMN were treated with TNF- Effects of ERK and p38 Inhibitors on the Activation of
Acetyltransferase--
To investigate the role(s) of the ERKs and p38
in acetyltransferase activation, we employed selective inhibitors
against p38 kinase, SB 203580, or MAP kinase kinase, PD 98059. These
drugs are highly selective for the respective kinases and do not appear to affect related protein kinases (20, 25-27). We (28) and others
(29-31) have shown that human PMN incubated with either 20 µM SB 203580 or 50 µM PD 98059 exhibits
~80% inhibition of their respective ERK and p38 kinase responses.
PMN were stimulated with TNF- Activation of Acetyltransferase by MAP Kinase(s) in Microsomal
Preparations--
To extend the findings from whole cells, we
established a cell-free system to test and further explore the
activation of the acetyltransferase by the MAP kinases. We employed
microsomes from unstimulated cells as our acetyltransferase source and
evaluated the effect of various recombinant, constitutively activated
MAP kinases on acetyltransferase activity. It has been recognized for
some time that the acetyltransferase is localized in an internal membrane source (32, 33), and although the compartment has not been
unequivocally determined, it has been suggested that it resides in
either the tertiary granule or endoplasmic reticulum membranes (34).
Regardless of the exact source, microsomal fractions were found to
contain the highest specific activity of acetyltransferase activity.
Microsomes (~5 µg of total protein) were incubated with
recombinant, constitutively activated ERK-1, ERK-2, or p38 kinase in
the presence of ATP and Mg2+. It should be noted that
Mg2+ dramatically inhibits acetyltransferase activity (12),
and as a result, only 0.5 mM Mg2+ was used in
the kinase assay. We have observed that this concentration of
Mg2+ has little to no effect on acetyltransferase activity
in our assay system (data not shown). Kinase reactions were allowed to incubate initially for 15 min, after which the entire mixture was added
to a reaction vessel containing acetyl-CoA, [3H]lyso-PAF,
and products assayed for acetyltransferase activity. We observed that
acetyltransferase activity was increased 2-3-fold in microsomes
isolated from neutrophils treated with TNF- Inactivation of Acetyltransferase in Microsomes by Treatment with
Alkaline Phosphatase--
To further establish if the activation of
acetyltransferase was because of a direct phosphorylation event,
membranes from control or TNF- Acetyl-CoA:lyso-PAF acetyltransferase has long been thought to be
regulated by a phosphorylation event; however, the specific kinase(s)
responsible for phosphorylation has yet to be unequivocally determined.
It has been suggested that protein kinase C (17), cAMP-dependent protein kinase (18), or
calcium/calmodulin-dependent protein kinases (19) could
activate acetyltransferase in vitro, but evidence for any
one of these kinases specifically activating the enzyme in whole cells
is lacking. Because acetyltransferase plays a critical role in
modulating the biosynthesis of PAF, understanding its regulation may
lead to novel therapeutic approaches.
In the present studies, we investigated three agents (TNF- Although we consistently observed no activation of acetyltransferase in
response to PMA, various reports have shown that PMA can either
activate or have no effect on acetyltransferase. Early reports by both
Albert and Snyder (13) in rat alveolar macrophages and by Domenech
et al. (19) in guinea pig exocrine cells clearly showed that
PMA treatment did not significantly activate acetyltransferase. Conversely, Lenihan and Lee (17) found that acetyltransferase activity
increased upon treatment of rat spleen microsomes with the soluble
fraction from rat spleen along with Mg2+ and ATP, and
because the activity could be further enhanced by the addition of
phosphatidylserine, diolein, and calcium, the authors speculated that
protein kinase C, the direct target of PMA, was playing a role (17). In
the neutrophil, no clear consensus has been reached as to whether
acetyltransferase is indeed activated by PMA. Our findings here and a
number of earlier findings (35, 36) indicate that PMA has little
measurable impact on acetyltransferase activity. These observations
disagree with those of Leyravaud et al. (37), who showed
that stimulation with PMA not only increased neutrophil
acetyltransferase activity but also raised PAF production. These
differences may reflect varying assay conditions including different
concentrations and times of incubations with PMA. Also, under certain
conditions, protein kinase C activation may be able to differentially
link to p38 activation.
For the purpose of our analyses, we selected relatively short
incubations of cells with high PMA concentrations. Under these conditions the phorbol ester achieved optimal activation of ERKs. We
were unable to find any concentration of PMA that appreciably activated
p38. In any event, other studies provided alternative explanations for
some of the observed differences. Nieto et al. (36) found
that PMA could in fact stimulate PAF production in PMN; however, the
PAF appeared to be synthesized de novo via the dithiothreitol-insensitive cholinephosphotransferase pathway. Because
PMA caused no activation of acetyltransferase, Nieto et al.
(36) accordingly concluded that PMA could only initiate PAF
biosynthesis through the de novo pathway. Similar results were later obtained in human umbilical vein endothelial cells where PMA
induced PAF formation solely through the de novo pathway (38). Although dithiothreitol-insensitive cholinephosphotransferase activity is high in PMN, the remodeling pathway appears to be the more
significant route by which PAF is synthesized in human PMN (39).
The formation of PAF is dependent upon at least two enzyme activities,
PLA2 and acetyltransferase. We have shown PMA can stimulate PMN to activate cPLA2, as well as to induce AA release when
measured by mass,2 so it appears that within the context of
the remodeling pathway, PMA can induce the generation of the obligate
PAF precursor, lyso-PAF, by activating PLA2, but the
acetylation of lyso-PAF does not occur at appreciable levels in
response to the phorbol ester. Our results suggest one possible
explanation for this relationship; acetyltransferase tightly controls
the formation of PAF. Alternatively, the lyso-PAF precursor could be
generated at a distinct site within the cell not accessible to acetyltransferase.
The p38 kinase has largely been considered to be a stress-activated
protein kinase in mammalian cells, as initial reports indicated that it
was activated in cells exposed to hyperosmolality, UV radiation, and
endotoxin (40, 41). More recently, other activators of p38 that act
through heterotrimeric G-protein-coupled receptors have been identified
that include interluekin-8 (42), fMLP, and PAF (43). PAF itself is
known to stimulate PAF biosynthesis in neutrophils (44) and other cell
types (15), possibly through the activation of acetyltransferase
directly. These observations agree with our findings, and we further
conclude that the regulation of acetyltransferase occurs via a direct
phosphorylation by p38. Although a complete definition of all the
signaling components activated in response to PAF has not been
elucidated, stimulation of neutrophils by PAF appears to trigger MAP
kinase kinase-3 activation, and evidence suggests that this occurs
through a pertussis toxin-insensitive pathway, possibly coupling to
G2 In conclusion, our results strongly indicate that p38 activates the
acetyltransferase responsible for PAF biosynthesis both in intact
neutrophils and cell-free preparations. The precise effect of the
phosphorylation event on subsequent PAF formation is still unclear,
because the activation of p38 impacts other enzymes involved in the
biosynthesis of PAF, i.e. cPLA2 (29, 31). The
determination of the specific enzyme, which may control the overall
flux through the PAF biosynthetic pathway, remains to be determined.
Our findings lead us to conclude that protein kinase C does not
activate the acetyltransferase in intact neutrophils. The findings fit
a model in which both cPLA2 (30, 32) and acetyltransferase are
activated in concert by the p38 kinase cascade in response to TNF- (TNF-
) as well as
N-formyl-methionyl-leucyl-phenylalanine treatment leads to
increased phosphorylation and activation of p38 kinase in human
neutrophils. Strikingly, in the present study these stimuli increased
the catalytic activity of acetyltransferase up to 3-fold, whereas
4-phorbol 12-myristate 13-acetate, which activates the
extracellular-regulated kinases (ERKs) but not p38 kinase, had no
effect. Furthermore, a selective inhibitor of p38 kinase, SB 203580, was able to abolish the TNF-
- and
N-formyl-methionyl-leucyl-phenylalanine-induced activation
of acetyltransferase. The same effect was not observed in the presence
of an inhibitor that blocked ERK activation (PD 98059). Complementing
the findings in intact cells, we have shown that recombinant, activated
p38 kinase added to microsomes in the presence of Mg2+ and
ATP increased acetyltransferase activity to the same degree as in
microsomes obtained from TNF-
-stimulated cells. No activation of
acetyltransferase occurred upon treatment of microsomes with either recombinant, activated ERK-1 or ERK-2. Finally, the increases in
acetyltransferase activity induced by TNF-
could be ablated by
treating the microsomes with alkaline phosphatase. Thus
acetyltransferase appears to be a downstream target for p38
kinase but not ERKs. These data from whole cells as well as cell-free
systems fit a model wherein stimulus-induced acetyltransferase
activation is mediated by a phosphorylation event catalyzed directly by
p38 kinase.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
was from PeproTech, Inc. (Rocky
Hill, NJ). Phorbol 12-myristate 13-acetate (PMA) was purchased from LC
Laboratories (Woburn, MA). Calf intestinal alkaline phosphatase was
from Promega (Madison, WI). Dulbecco's phosphate-buffered saline,
essentially lipid-free bovine serum albumin,
N-formyl-methionyl-leucyl-phenylalanine (fMLP), pepstatin A,
leupeptin, and phenylmethylsulfonyl fluoride were obtained from Sigma.
for 10 min. Treatments were terminated by the
addition of 30 ml of ice-cold phosphate-buffered saline without
CaCl2, and cells were pelleted at 300 × g
for 10 min at 4 °C. Pelleted cells were resuspended in 1 ml of
protection buffer (see above) and sonicated twice for 8 s at a
power setting of 2 and 10% output with a probe sonicator (Heat System
Inc.). Sonicates were centrifuged at 16,000 × g at
4 °C for 10 min to remove nuclei, granules, and cellular debris.
Supernatant fluids were further centrifuged in an airfuge (Beckman
Instruments) at 178,000 × g for 20 min at 25 °C.
The membrane fractions were resuspended in 500 µl of 0.2 M Tris-HCl (pH 7.5). Protein concentrations were determined
according to the method of Bradford (24) using bovine serum albumin as
the standard.
-treated cells
were incubated in the same manner except no protein kinases were added.
(1 nM) were incubated at 25 °C with 5 units of calf intestinal alkaline phosphatase in the
presence of assay buffer (50 mM Tris (pH 9.3), 1 mM MgCl2, 100 µM
ZnCl2, 1 mM spermadine) in a final volume of
100 µl. After 15 min, the entire 100-µl reaction mixture was added
to a reaction vessel containing acetyl-CoA (100 µM) and
[3H]lyso-PAF (16 µM, 0.1 µCi/tube) in 900 µl of 0.2 M Tris-HCl (pH 7.5) and shaken at 37 °C. The
reaction mixtures were extracted after 15 min and analyzed for
[3H]PAF formation as described above.
RESULTS
, PMA,
and Chemotactic Factors--
Previously, we investigated the abilities
of selected agents (TNF-
, PMA, and chemotactic factors) to activate
MAP kinase(s) in neutrophils and evaluated their subsequent impact on
cPLA2 activation.2 We found that
although all the agents tested activated cPLA2, there were
differences with regard to which MAP kinase cascade was utilized by the
individual stimuli. TNF-
and PMA were shown to preferentially
activate p38 kinase and ERKs, respectively. Further, it was shown that
both pathways were activated by chemotactic factors (i.e.
fMLP, C5a, and interleukin-8).
(1 nM, 10 min), PMA (10 nM, 10 min), or fMLP (100 nM, 1 min), sonicated, and assayed to determine
acetyltransferase activity (Fig. 1). We
observed that treatment with TNF-
induced a 2-3-fold increase in
acetyltransferase activity. Activation of acetyltransferase by fMLP was
also consistently observed, although the enzyme was activated to a
lesser degree. In contrast, there was no apparent activation of
acetyltransferase upon treatment of neutrophils with PMA. Although
baseline acetyltransferase activity varied from donor to donor, this
general trend remained constant throughout our studies.
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Fig. 1.
Activation of acetyltransferase by various
stimuli in intact human PMN. PMN (3 × 107) were
stimulated with 1 nM TNF- , 10 nM PMA, or 100 nM fMLP for 10, 10, or 1 min, respectively, or untreated
for 10 min (control). Cells were sonicated and subsequently
assayed for acetyltransferase activity as described under
"Experimental Procedures." Data represent the mean ± S.E. of
six separate experiments using different donors.
in the presence or absence of 20 µM SB 203580 or 50 µM PD 98059, either
alone or in combination. We observed that TNF-
-induced
acetyltransferase activity was almost totally abolished in the presence
of the p38 blocker, SB 203580, but was not affected by the inhibitor of
ERK activation, PD 98059 (Fig. 2).
Additionally, treatment with both drugs together resulted in no further
change compared with SB 203580 treatment alone. Albeit muted, similar
response patterns were seen in PMN treated with fMLP (Fig. 2). fMLP
alone induced a smaller but reproducible increase in acetyltransferase
activity. We again observed that blockage of p38 by SB 203580 resulted
in a loss of stimulated acetyltransferase activity whereas the
mitogen-activated protein kinase kinase inhibitor had no effect, either
alone or in combination with the p38 inhibitor.
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Fig. 2.
Inhibition of TNF- -
and fMLP-induced acetyltransferase activity in intact human PMN.
PMN (3 × 107) were incubated with or without 20 µM SB 203580 and/or PD 98059 for 30 min and subsequently
stimulated with 1 nM TNF-
or 100 nM fMLP for
10 or 1 min, respectively. Cells were sonicated and subsequently
assayed for acetyltransferase activity as described under
"Experimental Procedures." Data represent the mean ± S.E. of
four separate experiments using different donors.
, confirming our earlier
results with whole cell sonicates. Treatment with Mg2+ and
ATP alone effected no change in acetyltransferase activity, but the
addition of recombinant, constitutively activated p38 (2 µg) resulted
in a dramatic increase in activity, reaching levels observed in
microsomal membranes from TNF-
-treated cells (Fig. 3). This increase was not observed when
equivalent amounts (2 µg) of ERK-1 or ERK-2 were added. 3-Fold more
protein kinase was tested (6 µg), and no further increases in
acetyltransferase activity were observed, indicating that 2 µg of p38
gave full activation whereas even at higher levels, ERKs were still
inactive (data not shown). These data indicated that p38, not the ERKs,
is responsible for the phosphorylation and activation of
acetyltransferase in human neutrophils.
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Fig. 3.
In vitro activation of microsomal
acetyltransferase by recombinant, activated p38 kinase. Freshly
isolated membranes (40 µl, ~5 µg of total protein) were incubated
at 37 °C in the presence of 500 µM Mg2+
and 40 µM ATP in a final volume of 50 µl (complete
system) with 2 µg of recombinant, activated p38, ERK-1, or ERK-2.
Deletion of ATP/Mg2+ and additions of the kinases to the
complete system are indicated. After 15 min, the entire 50-µl kinase
reaction was added to a separate reaction vessel and subsequently
assayed for acetyltransferase activity as described under
"Experimental Procedures." As a positive control, microsomes from
TNF- -treated neutrophils were incubated in the same manner except no
protein kinases were added (TNF-
control). Data represent
the mean ± S.E. of four separate experiments using different
donors.
-stimulated PMN were treated with
alkaline phosphatase (Table I). Although
the specific activity from unstimulated cells was higher than routinely
observed, TNF-
still induced a 2-3-fold increase in
acetyltransferase activity. Upon treatment with alkaline phosphatase,
isolated membranes exhibited approximately 3-fold lower activity in
control and 6-fold lower activity in stimulated preparations. In fact,
phosphatase treatment reduced the activity of either treated or
untreated membranes to the same level (0.234 nmol/µg/15 min). These
results indicate that a phosphorylation event is crucial to not only
the activation of acetyltransferase but also to its basal level of
activation as assayed in stimulated or resting PMN, respectively.
Effect of alkaline phosphatase treatment on acetyltransferase activity
for 10 min at 37 °C; reactions were stopped,
and membrane fractions were prepared. Microsomal protein (5 µg) was
treated with (+) or without (
) 5 units of alkaline phosphatase for 15 min at 25 °C, and the membranes were subsequently assayed for
acetyltranferase activity as described under "Experimental
Procedures." Data represent the mean ± S.E. of three
experiments from different donors.
DISCUSSION
, PMA, and
fMLP) as potential activators of acetyltransferase in human
neutrophils. We observed that the best activator of p38, TNF-
, was
in fact the best activator of acetyltransferase. fMLP, which also
activated p38, albeit to a lesser degree, correspondingly activated
acetyltransferase to a lesser degree when compared with TNF-
. On the
other hand, PMA had virtually no effect on p38 or acetyltransferase
activity. The role of p38 in the activation of acetyltransferase was
further suggested in studies of intact neutrophils in which the p38
selective inhibitor (SB 203580) blocked acetyltransferase activation,
whereas the ERK inhibitor (PD 98059) did not. Next we were able to
activate the acetyltransferase in microsomal preparations by adding a
phosphorylating system containing constitutively active p38;
correspondingly, constitutively active ERK failed to activate the
acetyltransferase. Finally, the acetyltransferase activated by p38
could be inactivated by treatment with alkaline phosphatase. These
findings fully support activation of acetyltransferase via
phosphorylation of the enzyme by p38.
q or G
11 (43).
and other cytokines.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI-17287, HL-50395, and HL-56710.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Pharmacology and Cancer Biology, Duke
University Medical Center, Durham, NC 27710-3686. Tel.: 919-613-8614; Fax: 919-613-8642.
¶ To whom correspondence should be addressed. Tel.: 336-716-4372; Fax: 336-716-7671.
2 A. B. Nixon, J. T. O'Flaherty, L. M. S. Baker, and R. L. Wykle, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
PAF, platelet-activating factor;
PLA2, phospholipase
A2;
AA, arachidonic acid;
GPC, sn-glycero-3-phosphocholine;
PMN, polymorphonuclear neutrophil(s);
MAP, mitogen-activated protein;
PMA, 4-phorbol
12-myristate 13-acetate;
ERK, extracellular-regulated protein kinase;
TNF-, tumor necrosis factor-
;
fMLP, N-formyl-methionyl-leucyl-phenylalanine;
cPLA2, cytosolic PLA2.
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REFERENCES |
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