From the Departments of Biochemistry and
Medicine, Division of Infectious Diseases, Wake Forest
University School of Medicine,
Winston-Salem, North Carolina 27157
Received for publication, July 24, 2000, and in revised form, October 24, 2000
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ABSTRACT |
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In human neutrophils, the activation
of phospholipase D and the Tyr phosphorylation of proteins are early
signaling events upon cell stimulation. We found that the pretreatment
of neutrophils with ethanol (0.8%) or 1-butanol (0.3%), which results
in the accumulation of phosphatidylalcohol at the expense of
phosphatidic acid (PA), decreased the phorbol myristate
acetate-stimulated Tyr phosphorylation of endogenous proteins (42, 115 kDa). When neutrophil cytosol was incubated in the presence or absence
of PA, these and other endogenous proteins became Tyr-phosphorylated in
a PA-dependent manner. In contrast, phosphatidylalcohols exhibited only
25% (phosphatidylethanol) or 5% (phosphatidylbutanol) of the ability
of PA to stimulate Tyr phosphorylation in the cell-free assay.
Similarly, other phospholipids (phosphatidylcholine,
phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine,
phosphatidylinositol, polyphosphoinositides, and sphingosine
1-phosphate) showed little ability to stimulate Tyr phosphorylation.
These data suggest that PA can function as an intracellular regulator
of Tyr phosphorylating activity. Gel filtration chromatography of
leukocyte cytosol revealed a peak of PA-dependent Tyr
phosphorylating activity distinct from a previously described
PA-dependent phosphorylating activity (Waite, K. A.,
Wallin, R., Qualliotine-Mann, D., and McPhail, L. C. (1997) J. Biol. Chem. 272, 15569-15578). Among the protein
Tyr kinases expressed in neutrophils, only Fgr eluted exclusively in
the peak of PA-dependent Tyr phosphorylating activity.
Importantly, Fgr isolated from unstimulated neutrophil lysates showed
increased activity in the presence of PA but not phosphatidylbutanol.
Moreover, the pretreatment of neutrophils with 1-butanol decreased Fgr
activity in cells stimulated with formyl-methionyl-leucyl phenylalanine plus dihydrocytochalasin B. Together, these results suggest a new
second messenger role for PA in the regulation of Tyr phosphorylation.
Neutrophils are dynamic, short lived cells that play a vital role
in the early phases of the immune response. They provide formidable
killing mechanisms against invading organisms. Neutrophils sense their
environment through a variety of cell surface receptors that allow the
cells to respond to bacterial products, inflammatory cytokines,
immunoglobulins, complement cleavage products, and adhesion molecules.
Upon receptor ligation, numerous signaling events ensue within the cell
that culminate in a repertoire of functional responses appropriate for
a given stimulus. Among the many signaling events induced, increased
Tyr phosphorylation of proteins is commonly observed for a wide variety
of neutrophil stimuli (1, 2). Importantly, Tyr phosphorylation appears to play a central role in signaling pathways because treatment of
neutrophils with inhibitors of protein Tyr kinases has been shown to
interfere with most cellular functions (reviewed in Ref. 3).
Neutrophils express a large number of nonreceptor protein Tyr kinases
(2-4) with representatives from eight of the nine known families (5).
The functional roles of only a few of these enzymes in neutrophils have
been clearly defined (reviewed in Ref. 2). Similarly, little is known
about the regulation of protein Tyr kinases in neutrophils. The factors
that influence Tyr phosphorylation in neutrophils are diverse and may
reflect the variety of enzymes expressed in these cells. Examples of
signaling events shown to alter protein Tyr kinase activity include
ligand-induced dimerization of the Fc receptor (2), reactive oxygen
intermediates (4, 6, 7), and G protein activation (8). Recently, the
enhancement of Tyr phosphorylation by exogenously added (9-11) or
endogenously generated (9, 11)
PA,1 the lipid product of
phospholipase D, has been observed in neutrophils and other cell types.
The mechanism by which PA stimulates Tyr phosphorylation has not been addressed.
Like Tyr phosphorylation, the activation of phospholipase D is an early
cellular signaling event and has been linked to functional responses of
the neutrophil (12, 13). This enzyme metabolizes membrane
phospholipids, in a stimulus-dependent manner, yielding PA
and the free head group (e.g. choline or ethanolamine).
Although slow to gain acceptance as a lipid second messenger, PA has
now been reported to interact with or activate a variety of signaling components including protein Ser/Thr kinases (14-17), protein Ser/Thr (18) and Tyr (19) phosphatases, a cyclic nucleotide phosphodiesterase (20), regulators of G proteins (21, 22), lipid kinases (23), and
phospholipases (24, 25). Thus it is possible that the PA-stimulated Tyr
phosphorylation previously observed (9-11) could be due to the
interaction of PA with any of these signaling molecules or perhaps a
protein Tyr kinase. In this report, we examined the stimulatory role of
PA on Tyr phosphorylating activity in neutrophils, characterized this
activity, and identified at least one protein Tyr kinase that appears
to be regulated by PA. Our findings suggest a novel facet of protein
Tyr kinase regulation and a new signaling role for PA.
Materials--
Phorbol myristate acetate (PMA),
formyl-methionyl-leucyl phenylalanine (fMLP), dihydrocytochalasin B,
phosphatidylinositol 4-phosphate, and phosphatidylinositol
4,5-bisphosphate were obtained from Sigma. The anti-phospho-Tyr (clone
4G10) antibody was purchased from Upstate Biotechnology (Lake Placid,
NY). Fgr antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA;
for immunoprecipitation) and Wako Bioproducts (Richmond, VA; for
immunoblotting). Other protein Tyr kinase antibodies were obtained from
Santa Cruz Biotechnology (Lyn, Hck, Syk), Calbiochem (cAbl) and Dr.
Peter Greer (Queen's University, Canada; Fer, Fes).
Anti-p47phox (26) and Escherichia coli
containing the plasmid encoding glutathione S-transferase
(GST)-p47 fusion protein were gifts from Dr. Thomas Leto (National
Institutes of Health). The GST-p47 fusion protein was prepared as
described previously (27). Horseradish peroxidase-conjugated secondary
antibodies were from Transduction Laboratories (Lexington, KY). Protein
A-Sepharose beads were from Amersham Pharmacia Biotech. Enolase was
from Roche Molecular Biochemicals. The following lipids were purchased
from Avanti Polar Lipids (Alabaster, AL): the phosphatidic acids (PA)
dioctanoyl (di8:0), dicapryl (di10:0), dilauroyl (di12:0), dipalmitoyl
(di16:0), distearoyl (di18:0) and dioleoyl (di18:1); dioleoyl
phosphatidylethanol; dioleoyl phosphatidylbutanol; dioleoyl phosphatidylglycerol; and sn-1 oleoyl lyso-PA.
Phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol,
phosphatidylinositol, phosphatidylserine, and egg lecithin-derived PA
were obtained from Doosan Serdary Research Labs (Englewood Cliffs, NJ).
Phosphatidylinositol 3,4,5-trisphosphate and sphingosine 1-phosphate
were from Biomol (Plymouth Meeting, PA). Erbstatin analog, GF109203X,
herbimycin A, KN-62, and staurosporine were purchased from Alexis (San
Diego, CA). The following protein kinase inhibitors were from
Calbiochem: genistein, piceatannol, PP1, PD59058 and SB203580. C-I
(1-(5-isoquinolinesulfonyl) piperazine) was synthesized (28) by Dr.
Michael Thomas (Wake Forest University School of Medicine).
[ Neutrophil/Leukocyte Isolation, Stimulation, and
Fractionation--
Neutrophils were isolated from heparinized venous
blood obtained from healthy adult volunteers by dextran (T500; Amersham Pharmacia Biotech: for unit volumes of blood) or Isolymph
(Gallard-Schlesinger Industries, Carle Place, NY; for small volumes of
blood) sedimentation, centrifugation over Isolymph, and hypotonic lysis
of contaminating erythrocytes as described previously (29). Pooled
leukocytes were isolated from whole blood as described for neutrophils
except that the Isolymph centrifugation step was omitted. All
neutrophil (>95% purity) or leukocyte preparations were resuspended
at 5 × 107 cells/ml in cold Hanks' buffered salt
solution (Life Technologies, Inc.) and treated with diisopropyl
fluorophosphate (DFP) as described previously (30). For cell
stimulation experiments, neutrophils (5 × 106
cells/ml) were warmed for 5 min at 37 °C in the presence or absence of ethanol (0.8%, v/v) or butanol (0.3%, v/v) and then stimulated (10 nM PMA or 1 µM fMLP plus 10 µM
dihydrocytochalasin B) for various times. The incubation was terminated
by collecting cells after a rapid centrifugation (30-40 s, 9500 × g). In experiments designed to assess whole cell Tyr
phosphorylation (Fig. 1), cells were rapidly resuspended in ice-cold
protection buffer (phosphate-buffered saline plus 10 mM
EDTA, 5 mM EGTA, 25 mM NaF, 5 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, 1 mM DFP, 10 µg/ml leupeptin, 10 µg/ml
pepstatin and 1 µg/ml aprotinin) (31). An equal volume of 2× Laemmli
sample buffer (32) was added, and the cell lysate was immediately
boiled for 10 min. Aliquots (4.5 × 105 cell
equivalents) of lysates were subjected to SDS-PAGE and immunoblotting for phospho-Tyr detection, described below. In experiments designed to
assess Fgr activation, pelleted cells were lysed in ice-cold solubilization buffer (10 mM Tris (pH 7.5), 100 mM NaCl, 2 mM EGTA, 2 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 2 µg/ml pepstatin, 10 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM DFP, 1%
Nonidet P-40) for 15 min on ice with 10 passages through a 28-gauge
needle. The lysate was cleared by centrifugation and subjected to
immunoprecipitation. Neutrophil cytosol was obtained from cells
disrupted by sonication. The resultant lysate was subjected to
ultracentrifugation over a discontinuous sucrose gradient (15/40%,
w/v) as described previously (30, 33). The supernatant of pooled
leukocyte lysate subjected to ultracentrifugation (60 min, 4 °C,
150,000 × g) in the absence of the sucrose gradient
was collected as the cytosol. All cytosolic fractions were stored at
Preparation of Lipids--
Lipids were prepared daily by drying
a known volume of chloroform stock solution under a stream of nitrogen
and then sonicating in deionized water with a probe sonicator. The
concentration of phospholipids was verified by lipid phosphorus
analysis (34).
Tyr Phosphorylation Assay--
The cell-free Tyr phosphorylation
assay was carried out in kinase buffer (50 mM
NaPO4 buffer (pH 7), 1 mM EGTA, 5 mM MgCl2, 0.1 mM
Na3VO4) plus 100 µM lipid, 30 µM ATP, 6.7 µg/ml exogenous substrate (GST-p47), and
cytosolic protein (0.1 mg/ml) in a final volume of 135 µl. The
reaction mixture (minus cytosol) was warmed for 5 min at 25 °C, and
cytosol was added and incubated for 45 min. The reaction was terminated
by the addition of 15 µl of 10× dithiothreitol in Laemmli sample
buffer. Samples were quickly boiled and subjected to SDS-PAGE. In
preliminary experiments, phosphatidylcholine was found ineffective at
stimulating Tyr phosphorylation, but it allowed for exogenous substrate
recovery on immunoblots to an extent equal to that of PA. Thus,
phosphatidylcholine was routinely used in the minus PA control condition.
The phosphorylation assay was modified for the measurement of kinase
activities in gel filtration column fractions (Fig. 5) to compare
protein Tyr kinase (detected by anti-phospho-Tyr immunoblotting) and
protein Ser/Thr kinase (detected by incorporation of radiolabel) activities in parallel. The ATP concentration was reduced to 8 µM, and gelatin (0.1 mg/ml), an inert bulk protein, was
included to ensure equivalent recovery of exogenous substrate protein
on immunoblots. The protein Tyr kinase activity was determined in the
presence of Na3VO4 and unlabeled ATP, whereas
protein Ser/Thr kinase activity was determined in the absence of
Na3VO4 and radiolabeled ATP (10 Ci/mmol, (16)).
Preliminary experiments indicated that Tyr phosphorylation was not
readily detected at the reduced ATP concentration (8 µM)
in the absence of Na3VO4. In addition, at 8 µM ATP, there was a negligible increase in radiolabel
incorporation into the exogenous substrate in the presence of
Na3VO4 compared with its absence. This is
probably due to the large number of Ser (compared with Tyr)
phosphorylation sites in p47phox (35). Thus, the incorporation
of radiolabel into the exogenous substrate in the absence of
Na3VO4 was used to estimate PA-stimulated protein Ser/Thr kinase activity.
Immunoprecipitation and Immune Complex Kinase
Assay--
Neutrophil lysates were cleared with protein A-Sepharose
and rabbit IgG (30 min, 4 °C). Fgr was isolated by
immunoprecipitation by incubation with Fgr antibody and protein
A-Sepharose for 2 h (4 °C). Fgr-bound beads were washed three
times with solubilization buffer without DFP, EDTA, or Nonidet P-40 and
then twice with kinase buffer. The washed beads were prepared for the
immune complex kinase assay by the addition of 40 µl of kinase buffer
and warmed for 5 min at 25 °C before the addition of 10 µl of ATP
mix containing 10 µCi of [32P]ATP (6000 Ci/mmol).
Samples were incubated for 45 min at 25 °C, terminated by the
addition of 12.5 µl of 5× dithiothreitol in Laemmli sample buffer,
and prepared for SDS-PAGE.
SDS-PAGE and Immunoblotting--
Protein samples were
electrophoretically separated on 7 (phospho-Tyr) or 8% (Fgr activity)
gels and transferred to nitrocellulose in a transfer buffer containing
25 mM Tris-HCl, 192 mM glycine, 20% methanol,
supplemented with 0.1% SDS (36) to promote the efficient transfer of
the hydrophobic GST fusion protein exogenous substrate. Blots were
subjected to immunoblotting and visualized by enhanced
chemiluminescence (Pierce) as described previously (30), except that
blots probed for phospho-Tyr were blocked with 0.04% gelatin plus
0.5% goat serum in Tris-buffered saline with Tween 20 (Tris-buffered
saline, 10 mM Tris-HCl (pH 7.5), 100 mM NaCl,
0.1% Tween 20). To verify recovery of the exogenous substrate, blots
were stripped (Chemicon, Temecula, CA) and reprobed with the
anti-p47phox antibody. Tyr phosphorylation data for the
exogenous substrate were routinely normalized to exogenous substrate
protein to account for small differences in sample loading. Immunoblots
were analyzed and quantified by densitometry (PDI, Huntington Station,
NY); data from radiolabeled kinase assays were quantified by
PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA).
Gel Filtration Chromatography--
Leukocyte cytosol (~130 mg
of protein) was fractionated with ammonium sulfate (40%, v/v, final
concentration) and prepared for gel filtration chromatography on a
Sephacryl S-200 column exactly as described previously (16). Fractions
(4 ml, flow rate = 2 ml/min) were collected and assayed for
PA-dependent kinase activity using the modified kinase
assay described above. The column was calibrated with the following
molecular mass standards (Sigma) loaded and eluted individually:
thyroglobulin (669 kDa, void), ATP Determination--
ATP was measured in neutralized
perchloric acid extracts of Tyr phosphorylation assay reaction mixtures
using the luciferin-luciferase method according to the manufacturer's
protocol (Molecular Probes, Eugene, OR).
Pretreatment of Neutrophils with Primary Alcohols Reduces
PMA-stimulated Tyr Phosphorylation--
PMA has been shown to
stimulate both Tyr phosphorylation (37-39) and PA production (40) in
neutrophils. To determine whether PMA-stimulated Tyr phosphorylation is
related to PA production, we pretreated neutrophils with ethanol or
1-butanol, both commonly used tools to study phospholipase D-derived
PA. These primary alcohols act by competing with water as the hydroxyl
donor in the hydrolysis of membrane phospholipids by phospholipase D
(41). Thus, in the presence of primary alcohols, the
phosphatidylalcohol is produced at the expense of PA (41, 42). Fig.
1A shows that PMA stimulated
the Tyr phosphorylation (detected by immunoblotting) of many neutrophil
proteins (compare lanes 1 versus 3 and
5 versus 6), whereas others were constitutively
phosphorylated or dephosphorylated. Two proteins (~115 and 42 kDa)
exhibited Tyr phosphorylation that was reduced by the pretreatment of
cells with either ethanol (lane 4) or 1-butanol (lane
7). In contrast, the secondary alcohol, 2-butanol (lane
8), had little effect on the PMA-stimulated Tyr phosphorylation of
these proteins. Since primary, but not secondary, alcohols can be
utilized by phospholipase D, PA levels in 2-butanol-treated cells are
not compromised.
When data from several experiments were pooled, we found that the
PMA-stimulated Tyr phosphorylation of these proteins (~115 and 42 kDa) was transient but reached a maximum by 5 min (Fig. 1, B
and C). The PMA stimulation of PA accumulation in
neutrophils occurs over a similar time course (40). Moreover, the Tyr
phosphorylation of these proteins at 5 min was reproducibly reduced by
ethanol pretreatment of cells. Taken together, these findings suggest that PA may play a role in enhancing Tyr phosphorylation in stimulated neutrophils.
Neutrophil Cytosol Contains PA-stimulated Tyr Phosphorylating
Activity--
To characterize the PA-stimulated Tyr phosphorylating
activity in neutrophils, we developed a cell-free assay to measure this activity. Fig. 2A shows a
typical phospho-Tyr immunoblot (right panel) of neutrophil
cytosol that was incubated in the presence or absence of 100 µM dicapryl (di10:0) PA. In the absence of PA, only
proteins in the 50-60-kDa range became Tyr-phosphorylated. In
contrast, the presence of PA resulted in the Tyr phosphorylation of
many endogenous proteins ranging in size from ~30 to 205 kDa (right panel, open arrowheads). Importantly, proteins that
were Tyr-phosphorylated in intact neutrophils in an alcohol-sensitive manner (115 and 42 kDa; Fig. 1) were also
Tyr-phosphorylated in a PA-dependent fashion in the
cell-free assay. This suggests that the cell-free Tyr-phosphorylating
assay is potentially useful in defining substrates of potential
PA-regulated protein Tyr kinase as well as the PA target(s) responsible
for the Tyr phosphorylation.
Fig. 2A also shows that an exogenous substrate, the GST
fusion protein of p47phox (GST-p47, filled
arrowhead), was Tyr-phosphorylated in a PA-dependent manner. The difference in the phosphorylation status of the exogenous substrate in the two incubation conditions was not due to differences in the amount of substrate protein. The phospho-Tyr blot was stripped and reprobed with an anti-p47phox antibody to confirm that the
amount of substrate was the same in both incubation conditions (Fig.
2A, left panel). Employing this exogenous substrate
facilitated the comparison of Tyr phosphorylating activity in different
cytosol preparations and quantification of the data. An examination of
other exogenous substrates showed that recombinant p47phox
(produced in a baculovirus system (26)) and enolase, to a lesser extent, also became Tyr-phosphorylated in the presence of PA (not shown). In contrast, other substrates used to measure protein Tyr
kinase activity (poly(Glu, Tyr)1:1 and poly(Glu,
Tyr)4:1) were not Tyr-phosphorylated (not shown).
The Tyr phosphorylation of substrates occurred in a
time-dependent fashion. When reactions were incubated at
25 °C, the PA-dependent Tyr phosphorylation of the
exogenous substrate increased linearly for about 45 min and then
plateaued (Fig. 2B). In contrast, the PA-dependent Tyr phosphorylation of p115 occurred more
rapidly. In the absence of PA, Tyr phosphorylation of substrates was
negligible (not shown).
To characterize further PA-stimulated Tyr phosphorylation, we assessed
the pH and divalent cation requirements. The pH maximum for
PA-dependent Tyr phosphorylation was between pH 6.5 and 7.0 for both the exogenous substrate and p115 (not shown). We chose to use
pH 7.0 in our standard assay conditions. With respect to divalent
cations, we found that the PA-dependent Tyr phosphorylation was not different in the presence of Mg2+ and
Mn2+ (99 ± 3% of Mg2+ control; mean ± S.E., n = 3; not shown) in Tris-buffered reaction mixtures (not shown). Tyr phosphorylating activity could not be measured in NaPo4-buffered reaction mixtures, because a
manganese salt precipitated. Finally, the addition of Ca2+
(1 mM) to either Mg2+- or
Mn2+-containing reaction mixtures resulted in an
approximate 50% decrease in the PA-dependent Tyr
phosphorylation of the exogenous substrate, whereas the Tyr
phosphorylation of endogenous proteins was almost completely blocked
(not shown).
Lipid Specificity of Stimulated Tyr Phosphorylation--
Many
studies have shown that the treatment of neutrophils with either
ethanol or 1-butanol results in the production of the corresponding
phosphatidylalcohol in the stimulated cell (11, 12, 43-46). Since this
treatment decreased PMA-stimulated Tyr phosphorylation in intact cells
(Fig. 1), phosphatidylalcohols should be less efficient activators of
Tyr phosphorylation than PA, if PA is involved in this response. To
test this hypothesis, we assessed the ability of phosphatidylethanol
and phosphatidylbutanol to mimic the ability of PA to stimulate Tyr
phosphorylation in the cell-free assay. Fig.
3 shows that phosphatidylethanol induced the Tyr phosphorylation of the p115 to only 25% (p < 0.001) of the level induced by PA having the same acyl chain
composition (dioleoyl, di18:1). This suggests that we probably
underestimated the PMA-stimulated Tyr phosphorylation of endogenous
proteins due to PA in intact cells pretreated with ethanol (Fig. 1,
B and C). The inability of phosphatidylbutanol to
stimulate the Tyr phosphorylation of endogenous protein was more
striking. The presence of phosphatidylbutanol stimulated only 5%
(p < 0.001) of the Tyr phosphorylation of p115
compared with the dioleoyl PA control conditions. In contrast to the
endogenous protein, we did not observe a marked difference between the
ability of phosphatidylethanol and phosphatidylbutanol to stimulate the
Tyr phosphorylation of the exogenous substrate, which exhibited only 34 and 42%, respectively, of the activity of PA.
As a positive control, we included dicapryl (di10:0) PA in the
experiments shown in Fig. 3. The data suggest that PAs of different acyl chain length may vary in their ability to stimulate Tyr
phosphorylation. We therefore examined the importance of acyl chain
composition on PA-stimulated Tyr phosphorylation using synthetic and
naturally occurring (derived from egg lecithin) PAs. Dioleoyl (di18:1)
PA was as effective as the shorter chain dicapryl PA in stimulating the
Tyr phosphorylation of p115 (Fig.
4A) and the exogenous
substrate (Fig. 4B). The naturally occurring egg
lecithin-derived PA (egg; at 100 µM) exhibited 53 and
75% of the Tyr phosphorylation stimulating activity of dicapryl PA for
p115 (Fig. 4A) and the exogenous substrate (Fig.
4B), respectively.
PAs with short (di8:0), intermediate (di12:0 and di16:0), and long
(di18:0) saturated acyl chains were further tested for their ability to
stimulate the Tyr phosphorylation of the exogenous substrate (Fig.
4C). Like dicapryl PA, the short chain dioctanoyl (di8:0) PA
was an effective activator of Tyr phosphorylation. However, as the
saturated acyl chain length increased, the ability of PA to stimulate
Tyr phosphorylation decreased. The presence of unsaturation in dioleoyl
(di18:1) PA restored the ability of the lipid to stimulate Tyr
phosphorylation. Whether the inability of the intermediate and long
saturated acyl chain PAs to activate the Tyr phosphorylation response
was due solely to chain length or to different physical properties of
the vesicles formed upon preparation of these lipids is not clear.
However, preparation of these PAs in phosphatidylcholine vesicles or in
Triton micelles did not improve their ability to stimulate the PA
response (not shown). Thus it appears as though PAs with at least one
long, unsaturated acyl chain (dioleoyl and egg lecithin-derived PA) preferentially stimulate the Tyr phosphorylation of the exogenous substrate and endogenous proteins. The short chain (8:0 and 10:0) PAs
may be of suitable chain length to mimic the proximal portion of the
acyl chains in dioleoyl (di18:1) PA, which has a site of unsaturation
at carbon 9 along the acyl chain. Together, these data suggest the acyl
chain composition of PA is an important determinant for interaction
with the lipid-sensitive target.
Cell membranes are composed of several phospholipids, and the
activation of neutrophils results in the generation of a variety of
lipid second messengers. Thus, it was important to determine whether
other lipids could mimic the ability of PA to stimulate Tyr
phosphorylation. Phospholipids were prepared in a uniform manner and
tested at a final concentration of 100 µM for their ability to stimulate the Tyr phosphorylation of the exogenous substrate
(Table I). PA, either naturally occurring
or synthetic, was clearly the most effective activator of exogenous
substrate Tyr phosphorylation. In contrast, the principal precursor of
PA in neutrophils, phosphatidylcholine, showed little stimulatory activity. Similarly, metabolites of PA (lyso-PA, diacylglycerol, and
glycerol phosphate) exhibited little or no ability to stimulate Tyr
phosphorylation of the exogenous substrate compared with PA. Likewise,
other membrane phospholipids, signaling lipids, and selected free fatty
acids were unable to mimic PA. The pattern of lipid-stimulated Tyr
phosphorylation of p115 was similar to that for the exogenous substrate
(not shown). These data indicate that the lipid-sensitive target(s)
responsible for increased Tyr phosphorylation has a preference for PA
and can discriminate among lipids.
PA-stimulated Tyr Phosphorylation Is Sensitive to Inhibitors of
Protein Kinases--
We next addressed the mode of action of PA.
Dialysis of neutrophil cytosol failed to alter PA-stimulated Tyr
phosphorylation compared with undialyzed cytosol (not shown). This
suggests that small cytosolic molecules (<10,000 Da) are not involved
in the lipid-stimulated response. Alternatively, the Tyr
phosphorylation observed in the presence of PA may only be an apparent
enhancement if ATP was consumed more rapidly in the absence of this
lipid, due to a protective effect of PA on ATP. This was not the case. At the end of the cell-free incubation, the ATP concentration had
decreased more in the presence of PA compared with its absence. Thus,
at an initial concentration of 30 µM ATP (used in the
cell-free kinase assay), there was a 30% decrease in the ATP
concentration in the presence of PA compared with a 25% decrease in
its absence (not shown). Similarly, there was a 50% decrease in the
ATP concentration in the presence of PA compared with 30% in its
absence when the initial concentration was 8 µM ATP (not
shown; used in the modified cell-free and immune complex kinase assays
below). This indicates that PA promotes ATP consumption.
The simplest explanation for PA-stimulated Tyr phosphorylation is
either the activation of a protein kinase or inhibition of a protein
phosphatase. The cell-free Tyr kinase assay was routinely performed in
the presence of Na3VO4, a protein Tyr
phosphatase inhibitor, and ATP was required to observe PA-stimulated
Tyr phosphorylation (not shown). These observations argue against the
involvement of protein Tyr phosphatase in the response. Since PA has
been reported to activate protein Ser/Thr kinases (14-17), we screened a number of protein kinase inhibitors to determine what type(s) of
enzymes was involved in the PA-stimulated response.
Neutrophil cytosol was pretreated for 5 min with inhibitors prior to
the addition of lipid, ATP, and exogenous substrate. Table
II indicates that inhibitors of both
protein Tyr and protein Ser/Thr kinases impaired the PA-stimulated Tyr
phosphorylation of the exogenous substrate. The results mirrored those
observed for the Tyr phosphorylation of the endogenous substrates (not shown). Compounds reported to be selective for protein Tyr kinases caused varied degrees of inhibition of the Tyr phosphorylation response, with genistein (47), PP1 (48), and piceatannol (49) being the
most effective (~30, 50, and 70% of control activity, respectively).
Herbimycin A and erbstatin analog, both commonly used protein Tyr
kinase inhibitors (50, 51), had only modest inhibitory effects on the
PA-dependent Tyr phosphorylation. Staurosporine, a potent
nonselective protein kinase inhibitor (52, 53), was the most effective
inhibitor of the PA-stimulated response.
Among the inhibitors of protein Ser/Thr kinases, the nonselective C-I
(28) caused a modest decrease in the Tyr phosphorylation response. In
contrast, GF109203X, a selective protein kinase C inhibitor (54), had
only a slight inhibitory effect. Surprisingly, inhibitors of
Ca2+/calmodulin kinase II (KN-62 (55)) and p38 (SB203580
(56)) reduced the PA-dependent Tyr phosphorylation of the
exogenous substrate to 20 and 30%, respectively, of control activity.
However, the calmodulin antagonist chlorpromazine (100 µM) had no effect on PA-stimulated Tyr phosphorylation
(not shown), and the assay routinely contained EGTA. These later
observations argue against a role for Ca2+/calmodulin
kinase II in the PA-stimulated response. Although present in
neutrophils, the p38 protein Ser/Thr kinase, a MAPK cascade component,
was not activated in our assay conditions, as judged by the inability
to detect the dual phosphorylated p38 by immunoblotting (not shown).
Thus, the inhibitors directed against Ca2+/calmodulin
kinase II and p38 appear to lack the selectivity originally attributed
to them, as noted by others for p38 (57). Finally, the inhibitor of the
dual specific protein kinase MAPK/extracellular signal-regulated kinase
kinase (PD98059 (58)) showed only a slight inhibitory effect on
PA-stimulated Tyr phosphorylation of the exogenous substrate,
suggesting that this protein kinase is probably not a major upstream
target of PA.
These data suggest that the PA-stimulated response is complex and may
involve a protein Ser/Thr kinase upstream of a protein Tyr kinase.
Alternatively, a lipid-activated protein Tyr kinase with unusual
sensitivities to protein kinase inhibitors provides the simplest
explanation to account for the observed results. Although the inhibitor
screen provided useful information, it did not clearly indicate the
nature of the PA target responsible for enhanced Tyr phosphorylation.
Identification of a PA-regulated Protein Tyr Kinase--
We have
previously shown that neutrophil cytosol contains a PA-activated
protein kinase with an approximate molecular mass of 125 kDa,
determined by gel filtration chromatography (16). The partially
purified enzyme preparation phosphorylated GST-p47 (the exogenous
substrate) on both Ser and Tyr residues, suggesting the presence of a
dual specific kinase or multiple protein kinases sensitive to PA. To
distinguish between these possibilities, we subjected leukocyte
(neutrophil + monocyte + lymphocyte) cytosol to gel filtration
chromatography and assayed the eluted fractions for both Tyr and Ser
phosphorylating activity. PA-stimulated phosphorylating activity was
comparable in cytosol from neutrophils, lymphocytes, and monocytes
(16).2 We therefore used
leukocyte cytosol to obtain sufficient starting material for
chromatographic studies. Fig. 5 shows
that the PA-dependent Ser/Thr phosphorylation (open
circle) of the exogenous substrate eluted in fractions
corresponding to a molecular mass of 108 ± 12 kDa (mean ± S.E., n = 3). This agrees with our earlier molecular mass estimation for the PA-activated protein kinase (16). In contrast,
the activity responsible for PA-dependent Tyr
phosphorylation (closed circle) of the same substrate eluted
with the void volume of the gel filtration column. These data clearly
indicate that the PA-stimulated Tyr- and Ser/Thr-directed
phosphorylation activities are distinct entities. Moreover, these
results show that the novel PA-stimulated protein Ser/Thr kinase (16)
is not a PA effector for PA-stimulated Tyr phosphorylation.
We next considered the possibility that PA could directly activate a
protein Tyr kinase. The elution fractions of the gel filtration column
were screened by immunoblotting for the presence of protein Tyr kinases
expressed in leukocytes. Of the protein Tyr kinases examined (cAbl,
Lyn, Hck, Fgr, Fes, Fer, and Syk), only Fgr was found to elute
exclusively in the peak of PA-stimulated Tyr phosphorylating activity
(Fig. 5, bottom panel). Although some of the other protein
Tyr kinases examined (cAbl, Lyn, Fes, and Fer) did elute in the peak of
PA-dependent Tyr phosphorylating activity, they were also
present in fractions that exhibited low activity (>fraction 28; not
shown). Hck and Syk eluted in fractions closely corresponding to their
estimated molecular masses (fractions 30-35; not shown).
These findings suggested that PA might activate Fgr. To test this
hypothesis, we took two approaches. First, we assessed the sensitivity
of Tyr phosphorylating activity in gel filtration column fractions to
PP1, the selective Src family inhibitor. We found that PP1 (100 nM) decreased PA-dependent Tyr phosphorylation in Fgr-containing fractions (fractions 22-24) by 28% (not
shown). Next, we isolated Fgr from lysates of unstimulated neutrophils by immunoprecipitation, and we assessed its kinase activity in the
presence of various lipids using an immune complex kinase assay. Fig.
6A shows that dioleoyl PA
stimulated a 2-fold increase in Fgr autophosphorylation compared with
the phosphatidylcholine (PC) control condition. This PA-induced
increase in Fgr activity was highly significant (p < 0.007). The use of an irrelevant antibody during immunoprecipitation
failed to isolate Fgr protein or a phosphorylated band comigrating with
Fgr (not shown). As an additional control, we added PP1 (100 nM) to immunoprecipitated Fgr, and we observed a blockade
of phosphorylating activity (not shown). In contrast to PA, the
presence of phosphatidylbutanol failed to stimulate Fgr
autophosphorylation. The autoradiographs below the graph show a
representative experiment of Fgr autophosphorylation (top)
and the corresponding Fgr protein (bottom) detected on the same blot. Thus, the differences in Fgr autophosphorylation were not
due to differences in amounts of kinase protein. Taken together, these
data provide evidence that PA can regulate the activity of a
nonreceptor protein Tyr kinase.
To determine whether a lipid regulatory mechanism of Fgr was operative
in intact cells, we stimulated neutrophils in the presence or absence
of butanol (0.3%), and the activity of immunoprecipitated Fgr was
assessed. Although many neutrophil agonists elevate intracellular PA,
we selected fMLP plus dihydrocytochalasin B as a stimulus since it is a
potent activator of phospholipase D (12). Moreover, fMLP/dihydrocytochalasin B-stimulated functional responses have been
shown to be sensitive to primary alcohols (12). When neutrophils were
pretreated with dihydrocytochalasin B and then stimulated with fMLP,
Fgr autophosphorylating activity increased nearly 4-fold over that in
unstimulated cells (Fig. 6B). This observation was confirmed
by immunoprecipitating Fgr from lysates of unstimulated and stimulated
neutrophils with an antibody directed against a different Fgr epitope
(not shown). Pretreatment of cells with 1-butanol, a primary alcohol
(0.3%), significantly (p < 0.007) inhibited
fMLP/dihydrocytochalasin B-stimulated Fgr activity by 50%. In
contrast, treatment of cells with the corresponding secondary alcohol
(2-butanol), which can not be utilized by phospholipase D, had no
inhibitory effect on stimulus-induced Fgr activity. These data clearly
suggest that Fgr activity in the intact cell can be regulated in a
phospholipase D-dependent manner.
Our understanding of the cellular roles of protein Tyr kinases is
rapidly expanding because of molecular techniques that allow for the
manipulation of protein expression. In contrast, our understanding of
the regulatory events leading to the activation and inactivation of
these signaling molecules in the intact cell lags behind. Our findings
show that Tyr phosphorylating activity in human neutrophils can be
regulated in a positive manner by a cell membrane-derived lipid. This
represents a novel facet of protein Tyr kinase regulation. Moreover, it
links protein Tyr kinase activation to stimulus-induced membrane lipid
metabolism, both of which are early signaling events in most cell types.
The pattern of Tyr-phosphorylated proteins observed with PMA
stimulation of neutrophils was similar to that described previously (11, 38, 39). Moreover, certain proteins appear to be
Tyr-phosphorylated in a manner sensitive to primary alcohols (Fig. 1).
Our observations in primary neutrophils are in agreement with those
made in HL-60 cells, a line with the capability to differentiate into
neutrophil-like cells (11). Together, these findings implicate
phospholipase D and its product, PA, in the regulation of the Tyr
phosphorylation response. In addition, exogenous PA has been shown to
stimulate Tyr phosphorylation of neutrophil proteins by a mechanism
that does not require its entry into cells (10). Thus, neutrophil function appears to be enhanced by PA, acting as either an
intracellular messenger or an inflammatory mediator (59).
Our focus was on PA as an intracellular messenger. By using a cell-free
system, we showed that PA stimulated Tyr phosphorylation (Fig. 2).
However, PA metabolites, membrane phospholipids, and other signaling
lipids failed to mimic this ability of PA (Table I). Importantly,
phosphatidylalcohols, which are generated at the expense of PA upon
incubation of cells with primary alcohols, also failed to mimic the
ability of PA to stimulate Tyr phosphorylation (Fig. 3). Thus, the
target(s) responsible for enhanced Tyr phosphorylation is (are) clearly
able to discriminate among lipids (Table I and Figs. 3 and 4). This
strengthens the link between the early signaling events of
phospholipase D activation and Tyr phosphorylation.
The activation of a protein Tyr kinase or the inhibition of a protein
Tyr phosphatase provides the most straightforward explanation for
PA-stimulated Tyr phosphorylation. The latter has not been reported,
but PA activation of a protein Tyr phosphatase (SHP-1) has been
observed (19). Since protein Tyr kinases can be activated by Tyr
dephosphorylation (60), this could explain our observations. However,
reported properties of PA-stimulated SHP-1 activity differ significantly from those observed for PA-stimulated Tyr
phosphorylation. SHP-1 activity was stimulated by PA as well as by
phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and
arachidonic acid and was inhibited by Mn2+ (19). In
contrast, these phospholipids were poor activators of Tyr
phosphorylation (Table I); arachidonic acid was inactive (Table I), and
Mn2+ easily replaced Mg2+ in our assay
conditions. Thus, it is unlikely that SHP-1 activation by PA is
responsible for the observed lipid-stimulated increase in Tyr
phosphorylation. In addition, PA-stimulated Tyr phosphorylation required the presence of ATP (not shown); a protein Tyr phosphatase inhibitor (Na3VO4) was routinely included in
the Tyr phosphorylation assay mixture; and protein Tyr kinase
inhibitors diminished PA-stimulated Tyr phosphorylation (Table II).
These observations argue against the involvement of a protein Tyr
phosphatase in the PA response.
Neutrophils express numerous protein Tyr kinases (2-4). There is
limited information on the interaction of nonreceptor protein Tyr
kinases with lipids. However, some of these enzymes localize to
membranes via a myristoylated amino terminus (60), and Lyn and Fyn,
both Src family protein Tyr kinases, have been reported to interact
with glycolipids (61-63). The activity of purified Src was previously
reported to be enhanced by acidic phospholipids (64). Indeed, our
observation that PA-stimulated Tyr phosphorylating activity is
sensitive to inhibitors of protein Tyr kinases (Table II) suggests that
a protein Tyr kinase is involved in the response. Among the more
selective inhibitors tested, PP1, a Src family protein Tyr kinase
inhibitor (48), blocked the PA-stimulated response by nearly 50%. This
suggests that a member(s) of this protein Tyr kinase family may
participate in the PA-stimulated response. We observed that Fgr, a Src
family enzyme, eluted exclusively in the peak of
PA-dependent Tyr phosphorylating activity obtained from the
gel filtration fractionation of leukocyte cytosol (Fig. 5). Moreover,
PA-dependent Tyr phosphorylating activity in Fgr-containing fractions was decreased by PP1. Since no other protein Tyr kinase examined exhibited a comparable elution profile, we pursued Fgr as a
candidate enzyme for regulation by PA. Fgr is primarily a membrane-associated protein (65, 66),2 yet we detected this
protein Tyr kinase in the cytosolic fraction of cells disrupted by
sonication. However, to maximize Fgr recovery from cells, we opted to
isolate the protein by immunoprecipitation from whole cell lysates. We
found that PA, but not phosphatidylbutanol, could activate Fgr (Fig.
6A). Furthermore, Fgr activation in the intact cell appears
to be regulated in part by PA (Fig. 6B). A variety of
stimuli (e.g. opsonized zymosan, fMLP, PMA, the calcium ionophore A23187) are known to both elevate PA levels (12, 40, 67) and
activate Fgr (66, 68, 69) in suspended neutrophils. These observations
suggest that stimulus-dependent Tyr phosphorylation of
proteins is mediated, in part, by PA and Fgr.
The unique elution pattern of Fgr led us to discover its sensitivity to
PA. However, we cannot rule out the possibility that another protein
Tyr kinase(s) is regulated by PA. Several observations suggest that Fgr
is not alone in its regulation by PA. These include the following: 1)
PA-dependent Tyr phosphorylating activity was present in
elution fractions in which Fgr was not present; 2) the selective Src
family protein kinase inhibitor did not completely abolish
PA-stimulated Tyr phosphorylating activity; and 3) other protein Tyr
kinases coeluted with Fgr in the peak of PA-dependent Tyr
phosphorylating activity.
The mechanism by which PA might regulate the activity of Fgr is not yet
clear. Fgr is a 55-58-kDa protein, yet it eluted from the gel
filtration column in the void volume (Fig. 5). This suggests that Fgr
probably associates with a protein complex. Both cellular and viral Fgr
have been reported to associate with cellular proteins (70-73). This
raises the possibility of an indirect action of PA on the activation of
Fgr, i.e. a known PA effector may be activated by PA, which
in turn activates Fgr. The sensitivity of the PA-stimulated Tyr
response to inhibitors of protein Ser/Thr kinases (Table II) suggests
this possibility as well. Based on the data presented, we can rule out
some of the known PA effectors that could potentially regulate Tyr
phosphorylating activity. It is unlikely that protein kinase C is an
upstream effector of a protein Tyr kinase in the cell-free system. The
selective inhibitor of this protein Ser/Thr kinase had little effect on
PA-stimulated Tyr phosphorylation (Table II), and the activators of
protein kinase C (calcium, diacylglycerol ± PA,
phosphatidylserine) were inhibitory or had no effect on PA-stimulated
Tyr phosphorylation in the cell-free system. However, in the intact
neutrophil, protein kinase C may play a role in the alcohol-sensitive
Tyr phosphorylation due to its participation in the regulation of
phospholipase D (74, 75).
Among the other PA effectors, our data clearly indicate that the novel
PA-activated protein Ser/Thr kinase present in neutrophil cytosol (16)
is not an upstream regulator of a protein Tyr kinase. Activity
associated with this protein kinase eluted from the gel filtration
column in fractions distinct from those exhibiting PA-stimulated Tyr
phosphorylation activity (Fig. 5). Finally, PKN, which is activated by
free fatty acids, can also be activated by PA (76). In our cell-free
assay, neither arachidonic acid nor oleic acid, both potent PKN
activators, had stimulatory effects on the Tyr phosphorylation (Table
II) of exogenous and endogenous substrates. Thus, it is unlikely that
PKN is involved in PA-stimulated Tyr phosphorylation. The data
presented do not allow us to rule out an involvement of two other
candidate PA effectors, Raf-1 and Pak (p21-activated kinase), which are
expressed in neutrophils (16). Experiments are underway to determine
the mechanism by which PA activates Fgr.
Fgr and a related Src family kinase, Hck, have been implicated in
integrin signaling and cell adhesion (reviewed in Ref. 2). The
development of a double knock-out mouse, deficient in both Fgr and Hck,
has been useful in demonstrating the requirement of these protein Tyr
kinases in adhesion-dependent functional responses (77,
78). Interestingly, ethanol, at concentrations comparable to those used
in this study, has been reported to decrease neutrophil adhesion in
both in situ and in vitro systems (79, 80). In
addition, ethanol has been shown to interfere with both oxidative and
nonoxidative killing mechanisms in stimulated neutrophils (81, 82). It
is tempting to speculate that an interference with
PA-dependent regulation of Fgr may compromise neutrophil adhesion and adhesion-dependent cellular functions.
In summary, our data indicate that human neutrophils contain Tyr
phosphorylating activity that is activated by PA in the intact cell as
well as in a cell-free system. Furthermore, we have identified Fgr as
at least one of the protein Tyr kinases expressed in neutrophils that
can be activated by PA. The lipid regulation of protein Tyr kinases
(whether by a direct or indirect mechanism) constitutes a novel
regulatory mechanism for this functionally important class of signaling components.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (10 and 6000 Ci/mmol) was obtained from NEN
Life Sciences. All other chemicals were of the highest quality available.
70 °C. Protein concentrations were determined with the
Coomassie-Plus protein assay (Pierce) using bovine serum albumin as a standard.
-amylase (200 kDa), alcohol
dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic
anhydrase (29 kDa), and cytochrome C (12.4 kDa).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (64K):
[in a new window]
Fig. 1.
PMA-stimulated Tyr phosphorylation in
neutrophils is reduced by alcohol pretreatment. A,
neutrophils (5 × 106 cells/ml) were pretreated with
buffer (lane 1, 5 and 6), ethanol (0.8%,
lane 2 and 4), 1-butanol (0.3%, lane
7), or 2-butanol (0.3%, lane 8) for 5 min at 37 °C
and then stimulated with PMA (10 nM) or vehicle
(Me2SO) control for 5 min. Cell lysates were obtained as
described under "Experimental Procedures" and subjected to SDS-PAGE
and immunoblotting with an anti-phospho-Tyr antibody. The migration of
molecular mass standards is indicated on the left and
right. Arrows show the migration of proteins
exhibiting alcohol-sensitive Tyr phosphorylation. The immunoblots shown
are representative of 3-4 experiments. B and C,
neutrophils were incubated for the indicated times without ( ) or
with 10 nM PMA after a 5-min preincubation with buffer
(
) or ethanol (0.8%,
). The Tyr phosphorylation of p115
(B) and p42 (C) was assessed by anti-phospho-Tyr
immunoblotting. Values are the mean ± S.E. for cell preparations
derived from four different donors. Ethanol pretreatment significantly
reduced Tyr phosphorylation where indicated (*, p < 0.05; **, p < 0.02).
View larger version (33K):
[in a new window]
Fig. 2.
PA stimulates Tyr phosphorylation in
neutrophil cytosol in a cell-free system. A, neutrophil
cytosol (0.1 mg/ml) was incubated with the exogenous substrate in the
presence of 100 µM phosphatidylcholine ( PA)
or dicapryl (di10:0) PA (+PA) for 45 min at 25 °C. The
reaction was terminated by the addition of 10× Laemmli sample buffer.
Proteins were separated by SDS-PAGE, and Tyr-phosphorylated proteins
were detected by immunoblotting (right panel). The
open arrowheads show endogenous cytosolic proteins that were
Tyr-phosphorylated in a PA-dependent manner. The exogenous
substrate was detected on the same blot after stripping and reprobing
with anti-p47phox (left panel). The closed
arrowhead shows the exogenous substrate (GST-p47). The migration
of molecular mass standards is indicated on the left. The
results shown are representative of 7-8 neutrophil cytosol
preparations derived from different donors. B, the time
course of PA-dependent Tyr phosphorylation of the exogenous
substrate (
) and p115 (
) in the cell-free assay was determined
from anti-phospho-Tyr immunoblots by densitometric analysis. Data are
the mean ± S.E. for cytosol preparations derived from three
different donors.
View larger version (28K):
[in a new window]
Fig. 3.
Phosphatidylalcohols can not mimic the
ability of PA to stimulate Tyr phosphorylation. Neutrophil cytosol
(0.1 mg/ml) was incubated in the presence of 100 µM
phosphatidylcholine, dicapryl (di10:0) PA, dioleoyl (di18:1) PA,
dioleoyl phosphatidylethanol (PEt), or dioleoyl
phosphatidylbutanol (PBt). Reactions were terminated and
processed as described under "Experimental Procedures" for the
cell-free assay. The Tyr phosphorylation of p115 and the exogenous
substrate were quantified by densitometry. Data are presented as the
percentage of the activity of dioleoyl PA for each substrate after
subtraction of background Tyr phosphorylation determined in the
presence of phosphatidylcholine. Data are the mean ± S.E. for
cytosol preparations derived from six different donors. The data are
significantly different (*, p < 0.001) from the
dioleoyl PA condition where indicated.
View larger version (18K):
[in a new window]
Fig. 4.
Acyl chain specificity of PA-stimulated Tyr
phosphorylation. A and B, neutrophil cytosol
(0.1 mg/ml) was incubated with the exogenous substrate and
phosphatidylcholine (control) or PAs of the indicated acyl chain
composition at the concentrations shown for 45 min at 25 °C;
reactions were stopped and processed as described under "Experimental
Procedures." The PAs used were synthetic ( , dicapryl, di10:0;
,
dioleoyl, di18:1) or naturally occurring (
, egg; derived from egg
lecithin). The Tyr phosphorylation of p115 (A) and the
exogenous substrate (B) were detected by immunoblotting and
quantified by densitometry. PA-dependent Tyr
phosphorylation was calculated as the difference in activities between
the PA and phosphatidylcholine. To facilitate a comparison among
lipids, data are presented as the percentage of
PA-dependent activity for 100 µM dicapryl PA
and are the mean ± S.E. for cytosol preparations derived from
three different donors. C, neutrophil cytosol was incubated
with the exogenous substrate and 100 µM
phosphatidylcholine or PAs of short (di8:0, di10:0), intermediate
(di12:0), or long (di16:0, di18:0, di18:1) acyl chain length. To allow
for a more direct comparison of the lipids tested, the data are
expressed as the percentage of PA-dependent activity for
100 µM dicapryl PA. The data are the mean ± S.E.
for cytosol preparations derived from 3 to 4 different donors.
Inability of phospholipids and PA metabolites to mimic PA stimulation
of Tyr phosphorylation
Effect of protein kinase inhibitors on PA-dependent Tyr
phosphorylation of the exogenous substrate by human neutrophil cytosol
View larger version (35K):
[in a new window]
Fig. 5.
Leukocyte cytosol contains at least two
PA-stimulated phosphorylating activities. Leukocyte cytosol
(~130 mg) was adjusted to 40% saturated ammonium sulfate (v/v). The
precipitated proteins were redissolved and chromatographed on a
Sephacryl S-200 gel filtration column. Aliquots (40 µl) of eluted
fractions were incubated in the presence or absence of 100 µM dicapryl (di10:0) PA, the exogenous substrate, and
either unlabeled ATP (Tyr phosphorylation) plus
Na3VO4 or radiolabeled ATP (Ser/Thr
phosphorylation) minus Na3VO4, according to the
modified phosphorylation assay described under "Experimental
Procedures." Immunoblots (Tyr phosphorylating activity) and
autoradiographs (Ser/Thr phosphorylating activity) were analyzed by
densitometry and PhosphorImager analysis, respectively.
PA-dependent protein kinase activity was calculated as the
difference in activities between the PA and PC ( PA)
incubation conditions and normalized to densitometric data for the
exogenous substrate protein. The data show the PA-dependent
Tyr (
) and Ser/Thr (
) phosphorylating activities. The
dashed line represents the protein elution profile
(A280). The arrows at the
top of the graph indicate the elution of molecular mass
standards. The Fgr elution profile was determined by preparing equal
volumes of column fractions for SDS-PAGE followed by immunoblotting for
Fgr (bottom panel). The horizontal bar marks
fractions 22-24 in which Fgr eluted. The data shown are representative
of three experiments using cytosol from different donors.
View larger version (32K):
[in a new window]
Fig. 6.
Fgr is activated in vitro
and in vivo in PA-dependent
manner. A, Fgr was immunoprecipitated from the lysates
of unstimulated neutrophils and assayed for activity in the immune
complex kinase assay as described under "Experimental Procedures."
The indicated lipids (100 µM) were added to the
immobilized kinase prior to the addition of radiolabeled ATP and
incubated for 45 min at 25 °C, and the reactions were stopped and
processed as described under "Experimental Procedures." The data
are presented as activity relative to that in the presence of PC and
are the mean ± S.E. (n = 3, 4) for lysates
obtained from the cells of different donors. The autoradiograph of Fgr
autophosphorylation (top panel) and immunoblot of Fgr
protein (bottom panel) show results from a representative
experiment. The data were significantly different (*, p < 0.007, for PA versus PC; **, p < 0.003, for phosphatidylbutanol versus PA) where indicated.
B, neutrophils were pretreated with 10 µM
dihydrocytochalasin B and either Hanks' buffered salt solution,
1-butanol (1-Bt, 0.3%, final concentration) or 2-butanol
(2-Bt, 0.3%) for 5 min at 37 °C followed by a 5-min
stimulation with Me2SO (control) or 1 µM
fMLP. Fgr was isolated from cell lysates and its activity was assessed
using the immune complex kinase assay as described under
"Experimental Procedures." No additional lipid was added to the
reaction mixture. The data were normalized to Fgr protein recovered in
the immunoprecipitate and are presented as activity relative to that in
unstimulated cells (control) and are the mean ± S.E.
(n = 4) for cells from different donors. The 1-butanol
treatment was significantly different (*, p < 0.007)
from the fMLP-stimulated condition.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Thomas Leto (National Institutes of Health) for gifts of the p47phox antibody and E. coli for production of GST-p47 fusion protein; Dr. Shari Orlicek (Wake Forest University School of Medicine) for the PP1, a protein Tyr kinase inhibitor, and cAbl antibody; Dr. Robert Wykle (Wake Forest University School of Medicine) for the protein kinase inhibitors SB203580 and PD098059; Dr. Joseph O'Flaherty (Wake Forest University School of Medicine) for sharing the antibodies against dual phosphorylated p38 and p38 protein; Dr. Giorgio Berton (University of Verona) for generously sharing anti-Fgr antibodies; Dr. Peter Greer (Queen's University) for the generous gift of antibodies against Fer and Fes; and Drs. Reidar Wallin and Mark Lively (Wake Forest University School of Medicine) for helpful advice and the use of chromatography equipment. We also thank Dianne Greene for technical expertise in lipid phosphorus analysis, Mary Ellenburg for help with preliminary experiments, and Dr. Debra Regier for thoughtful and stimulating discussions.
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Addendum |
---|
We have now observed that the magnitude of PA-stimulated Fgr activity is more variable than that shown (Fig. 6A). The variability appears to result from as yet undefined differences in lots of tissue-derived phosphatidylcholine. After testing three lots of phosphatidylcholine, we find that Fgr activity in the presence of PA is 1.45 ± 0.16 (mean ± S.E., n = 8, p = 0.015)-fold higher than that in the presence of phosphatidylcholine.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant 2R01 AI22564 (to L. C. M.) and Wake Forest University School of Medicine Venture Funds (to S. S.).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.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-2586; Fax: 336-716-7671; E-mail: ssergean@wfubmc.edu.
¶ Current address: CIHR Group on Cell and Molecular Biology of Lipids, Dept. of Biochemistry, 320 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G2R3, Canada.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M006571200
2 S. Sergeant, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: p47phox, 47 kDa phagocytic oxidase component; DFP, diisopropyl fluorophosphate, Me2SO, dimethyl sulfoxide; fMLP, formyl-methionyl-leucyl phenylalanine; GST, glutathione S-transferase; PA, phosphatidic acid; PMA, phorbol myristate acetate; PC, phosphatidylcholine; MAPK mitogen-activated protein kinase, PAGE, polyacrylamide gel electrophoresis.
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