(Received for publication, September 6, 1996, and in revised form, March 21, 1997)
From the Departments of Biochemistry and Medicine,
Divisions of
Infectious Diseases and ¶ Rheumatology,
Wake Forest University Medical Center,
Winston-Salem, North Carolina 27157
Phosphatidic acid (PA), generated
by phospholipase D activation, has been linked to the activation of the
neutrophil respiratory burst enzyme, NADPH oxidase; however, the
intracellular enzyme targets for PA remain unclear. We have recently
shown (McPhail, L. C., Qualliotine-Mann, D., and Waite, K. A. (1995)
Proc. Natl. Acad. Sci. U. S. A. 92, 7931-7935) that a
PA-activated protein kinase is involved in the activation of NADPH
oxidase in a cell-free system. This protein kinase phosphorylates
numerous endogenous proteins, including p47-phox, a
component of the NADPH oxidase complex. Phospholipids other than PA
were less effective at inducing endogenous protein phosphorylation.
Several of these endogenous substrates were also phosphorylated during
stimulation of intact cells by opsonized zymosan, an agonist that
induces phospholipase D activation. We sought to identify the
PA-activated protein kinase that phosphorylates p47-phox.
The PA-dependent protein kinase was shown to be cytosolic.
cis-Unsaturated fatty acids were poor inducers of protein
kinase activity, suggesting that the PA-activated protein kinase is not
a fatty acid-regulated protein kinase (e.g. protein kinase
N). Chromatographic techniques separated the PA-activated protein
kinase from a number of other protein kinases known to be activated by
PA or to phosphorylate p47-phox. These included isoforms of
protein kinase C, p21 (Cdc42/Rac)-activated protein kinase, and
mitogen-activated protein kinase. Gel filtration chromatography indicated that the protein kinase has an apparent molecular size of 125 kDa. Screening of cytosolic fractions from several cell types and rat
brain suggested the enzyme has widespread cell and tissue distribution.
The partially purified protein kinase was sensitive to the same protein
kinase inhibitors that diminished NADPH oxidase activation and was
independent of guanosine 5-3-O-(thio)triphosphate and
Ca2+. Phosphoamino acid analysis showed that serine and
tyrosine residues were phosphorylated on p47-phox by this
kinase(s). These data indicate that one or more potentially novel
protein kinases are targets for PA in neutrophils and other cell types.
Furthermore, a PA-activated protein kinase is likely to be an important
regulator of the neutrophil respiratory burst by phosphorylation of the NADPH oxidase component p47-phox.
Phospholipase D (PLD)1 is activated in a variety of cells by hormones and growth factors (1-3). This activation results in the generation of phosphatidic acid (PA), which can be further metabolized by PA phosphohydrolase to diacylglycerol (DG). The generation of PA by PLD in neutrophils has been linked, by us and others, to the activation of the respiratory burst enzyme, NADPH oxidase (2-7). The enzymes involved in the activation of the oxidase, which are downstream of the generation of PA, have not been identified. We have developed a cell-free activation system for NADPH oxidase, which utilizes PA and is phosphorylation-dependent (7, 8). The system requires the presence of both cellular membranes and cytosol, as well as the presence of DG. Since a variety of potential enzyme targets for PA are present, this system provides a means to identify these enzymes and their role in activation of the NADPH oxidase. We have recently shown that, during phosphorylation-dependent oxidase activation, PA, but not DG, induces phosphorylation of a wide range of proteins, in which the most prominent protein phosphorylated is the NADPH oxidase component p47-phox (phagocytic oxidase component) (8). This indicates that potential targets for PA, involved in NADPH oxidase activation, are protein kinases.
Several protein kinases have been demonstrated to be activated by PA in vitro. These include isoforms of protein kinase C (PKC) (9-13) as well as the recently described fatty acid-activated protein kinases (14-20). In addition, Raf-1 (a mitogen-activated kinase kinase kinase) has been shown to bind anionic phospholipids, including PA, and to translocate from cytosol to membrane in Madin-Darby canine kidney cells in response to PLD activation (21). In vitro studies have shown that PKC, mitogen-activated protein kinase (MAPK) and isoforms of the newly identified p21 (Cdc42/Rac)-activated protein kinase (PAK) are able to phosphorylate the NADPH oxidase component p47-phox (22-25). Neutrophils are known to contain p42 and p44 MAPK isoforms (26, 27) and PAK-1 and PAK-2 (24, 28, 29). Potentially, p42 and p44 MAPK isoforms could be activated as a consequence of Raf-1 activation by PA (21). PA is known to be generated in neutrophils under conditions in which these protein kinases are activated (5, 6, 30). PAK activation could be a consequence of PA-mediated activation of the small GTPase Rac (24, 28, 29, 31), through the inhibition of Rho-GDp dissociation inhibitor (32, 33). In fact, the production of PA from PLD activation has been linked to the activation of PAK isoforms in neutrophils (34, 35). Thus, while MAPK and PAK isoforms are not known to be activated directly by PA, they could be activated downstream of other protein targets of PA and ultimately be responsible for the phosphorylation of p47-phox.
We examined whether the PA-dependent protein kinase activity in human neutrophils was due to the activation of one of these known protein kinases. Our results indicate that this is not the case and that a potentially novel protein kinase, which phosphorylates p47-phox, may be the target for PA in this system. This protein kinase may therefore play a role in the activation of the NADPH oxidase.
Materials
Sf9-expressed recombinant p47-phox (36) and
Escherichia coli containing the plasmid encoding glutathione
S-transferase (GST)-p47-phox fusion protein were
generous gifts from Dr. Tom Leto (National Institutes of Health). The
GST-p47-phox fusion protein was prepared as described
previously (37). The PA used was
1,2-dicapryl-sn-glycero-3-phosphate and was obtained from
Avanti Polar Lipids (Alabaster, AL). All other phospholipids were from
Serdary Research Laboratories (Port Huron, MI). Lipids were prepared
for use by sonication (7). Arachidonic acid (AA) and oleic acid were
from Nu-Chek-Prep Inc. (Elysian, MN) and were dissolved in ethanol (7).
[-32P]ATP (1 mCi/ml) and
[32P]H3PO4 (10 mCi/ml) were from
DuPont NEN. Phosphoserine, phosphotyrosine, and phosphothreonine
standards were from Sigma. Phosphocellulose paper (P-81) was from
Whatman (Hillsboro, OR), and G-25-Sepharose was from Pharmacia
(Uppsala, Sweden). Reagents for sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) and Western blotting were obtained from
Bio-Rad. Antibodies were obtained from the following sources:
anti-PKC-
, anti-phosphotyrosine (4G10), and anti-MAPK (anti-rat MAPK
R2) from Upstate Biotechnology Inc. (Lake Placid, NY);
anti-PKC-
from Oxford Biomedical Research (Oxford,
MI); anti-PKC-
from Life Technologies, Inc.; anti-PKC-
and
baculovirus-expressed PKC isoform standards from Dr. David Burns
(Sphinx Pharmaceuticals, Durham, NC); anti-Raf-1 and anti-B-Raf from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-PAK-1 a generous
gift from Dr. Arie Abo (Onyx Pharmaceuticals, Richmond, CA) (28); and
anti-PAK-2 kindly provided by Dr. Gary Bokoch (Scripps Research
Institute) (24). Conjugated horseradish peroxidase secondary antibodies
were obtained from Transduction Labs (Lexington, KY). Rabbit IgG was
from Sigma. Enhanced chemiluminescence (ECL) reagents for Western blot
analysis were obtained from Amersham Corp. Staurosporine was from
Biomol (Plymouth Meeting, PA), GF-109203X was from LC Laboratories
(Woburn, MA), and 1-(5-isoquinolinesulfonyl)piperazine (C-I) was
synthesized by Dr. Mike Thomas (Bowman Gray School of Medicine) (38).
Cell lines were generous gifts from the following individuals at Bowman
Gray School of Medicine: HL-60 (promyelocytic leukemia, human) and
Madin-Darby canine kidney cells, Dr. Larry Daniel; THP-1 (monocyte,
human), Dr. Charles McCall; CFTL-15 (mast cell, murine), Dr. Mary Beth
Fasano; RAW 264.7 (monocyte-macrophage, murine), Dr. B. Moseley Waite;
HepG2 (hepatocellular carcinoma, human) and McA-Rh7777 (hepatoma, rat),
Dr. Greg Shellness; IMR-32 (neuroblastoma, human), Dr. Osvaldo Delbono.
Rat brain tissue was a gift from Drs. Susan Hutson and Carol Cunningham
(Bowman Gray School of Medicine).
Isolation of Blood Cells and Subcellular Fractionation
Platelets were derived from heparinized venous blood by
centrifugation (300 × g, 20 min, 25 °C) (39). The
supernatant, containing platelet-rich plasma, was then centrifuged
(2500 × g, 15 min, 4 °C) (39), and the pellet was
resuspended in Buffer A (50 mM NaxPO4,
(pH 7.0), 11% sucrose, 130 mM NaCl, 1 mM EGTA, and 0.5 mM phenylmethylsulfonyl fluoride) and processed as
described below. Neutrophils, monocytes, and lymphocytes were isolated
from heparinized venous blood obtained from consenting human donors by
dextran sedimentation followed by Isolymph (Gallard-Schlesinger Ind.,
Inc., Carle Place, NY) centrifugation (40). After the Isolymph
centrifugation, the interface, containing monocytes and lymphocytes,
was washed with RPMI (Life Technologies) and plated (41). Once the
monocytes had adhered, the nonadherent cells (lymphocytes) were
collected in Buffer A at 1 × 108 cells/ml. The
monocytes were scraped off the plates and resuspended at 1 × 108 cells/ml in Buffer A (41). Monocytes and lymphocytes
were then processed as described below. The pellet after Isolymph
centrifugation, containing neutrophils, was cleared of contaminating
red blood cells by hypotonic lysis (42, 43). Isolated neutrophils were suspended to a concentration of 1 × 108 cells/ml in
Buffer A. All cells to be analyzed were sonicated to ~90% breakage
(visualized by microscopy) and processed as follows. To prepare
cytosolic and membrane fractions, sonicates were freed of unbroken
cells and nuclei by centrifugation (200 × g, 10 min), loaded onto a 15-40% discontinuous sucrose gradient at a 2:1:1 (v/v/v) ratio and centrifuged at 150,000 × g (SW50
rotor, 30 min) (40). Cytosolic fractions were collected from the top
layer down to the 15% interface, and membrane fractions were collected from the 15-40% interface and the 40% sucrose layer. To prepare cytosolic fractions only, sonicates were centrifuged at 150,000 × g (Type 50 rotor, 90 min), and the resulting supernatant was collected. Fractions were stored at 70 °C. Protein was determined using the Coomassie Plus Protein protocol from Pierce, which is based
on the Bradford method (44). Bovine serum albumin was used as a
standard.
Phosphorylation Assays
Intact Cell AssayIsolated neutrophils were suspended to 1 × 108 cells/ml in loading buffer (10 mM Hepes (pH 7.2), 137 mM NaCl, 0.8 mM MgCl2, and 5.4 mM KCl). [32P]H3PO4 (1 mCi/ml) was added, and the mixture was incubated at room temperature for 90 min (45). Labeled neutrophils were centrifuged and resuspended at 1.5 × 108 cells/ml in stimulation buffer (10 mM Hepes (pH 7.2), 137 mM NaCl, 1.8 mM MgCl2, 5.4 mM KCl, and 0.5 mM CaCl2) (45) and stimulated with 10 mg/ml opsonized zymosan for 5 min (46). Stimulation reactions were terminated by the addition of a 10-fold excess of stimulation buffer and immediate centrifugation. Stimulated neutrophils were then resuspended in diisopropylfluorophosphate treatment buffer (stimulation buffer plus 0.5 mM phenylmethylsulfonyl fluoride, pH 7.2, 50 µM leupeptin, 5.4 mM Na3VO4, and 25 mM NaF) and incubated with 1 mM diisopropylfluorophosphate for 5 min (47) on ice, at which time a 10-fold excess of stimulation buffer was added and the cells were centrifuged. Cells were then resuspended in a modified sonication buffer (10 mM Pipes, 1 mM EGTA, 103 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, pH 7.2, 50 µM leupeptin, 5.4 mM Na3VO4, 25 mM NaF, and 11% sucrose) (45) and sonicated to ~90% breakage as described above. Unbroken cells and nuclei were removed by low speed centrifugation, and subcellular fractions were obtained as described above. Proteins were separated by 8-15% SDS-PAGE (40, 48), silver-stained (49), dried, and analyzed via autoradiography.
Cell-free Assay: Analysis by SDS-PAGEReaction mixtures
(150 µl total) contained 50 mM NaxPO4
(pH 7.0), 1 mM EGTA, 5 mM MgCl2 (7,
8) plus one of the following: a combination of the cytosolic and
membrane fractions (25:1 protein:protein ratio), the cytosolic fraction
only, the membrane fraction only, partially purified enzyme, or column
fractions. GST-p47-phox (1 µg/reaction) or recombinant
p47-phox (Rp47, 1 µg/reaction) were added to the mixtures
as indicated in the figure legends. Six µl of
[-32P]ATP were added, followed by the immediate
addition of activator, as indicated in the figure legends. This mixture
was allowed to incubate at 25 °C for 45 min. The reaction was then
terminated by the addition of Laemmli sample buffer and prepared for
SDS-PAGE analysis (48). Proteins were separated by 8-15% SDS-PAGE
(40), silver-stained (49), dried, and analyzed via autoradiography. Protein kinase inhibitors were added 5 min prior to the addition of
[
-32P]ATP. For samples that were analyzed by Western
blotting, reactions were performed as above with the following
modifications. Cold ATP (10 µM) was used in place of
radiolabeled ATP. Proteins were separated by 9% SDS-PAGE and then
transferred to polyvinylidene difluoride (50).
Reaction mixtures were the same as above, except that
[-32P]ATP, diluted to yield 2 × 106
cpm/reaction, was used (final ATP concentration is indicated in figure
legends), and samples were allowed to preincubate for 5 min at 25 °C
before the addition of sample material to be tested (51). After 30 min,
75% (112.5 µl) of the reaction mixture was transferred to Whatman
P-81 phosphocellulose squares; the squares were washed with 75 mM phosphoric acid and quantitated by Cerenkov counting
(52, 53). 1 µM GTP
S or 0.6 mM
CaCl2 were added to samples as indicated under
"Results." For the mixed lipid vesicle experiments, PA, PS, PE, and
PC were prepared separately by sonication (7). PE, PC, and PS were then
mixed together (final assay concentration: 25, 25, and 10 µM, respectively) with PA (final assay concentration: 0-300 µM). These mixtures were then used in the place of
PA in the reaction mixtures. All samples were assayed in duplicate. Conditions used were linear with respect to protein concentration and time of incubation.
Saturated Ammonium Sulfate Precipitation
Cytosol (2 mg of protein) was cleared by centrifugation at
10,000 × g and then adjusted with a solution of
saturated ammonium sulfate at 4 °C to yield 40% ammonium sulfate
saturation. This mixture was kept on ice for 10 min and was centrifuged
(10,000 × g, 4 °C, 10 min) to collect precipitated
protein. The resulting supernatant was adjusted with saturated ammonium
sulfate to yield 70% saturation and was kept on ice for 10 min, and
the precipitate was collected by centrifugation. Both the 40 and 70%
protein precipitates were resuspended in 500 µl of Buffer B (Buffer A
plus 2 mM dithiothreitol). These samples, plus the
resulting 70% supernatant, were cleared of residual ammonium sulfate
by passage over a Sephadex G-25 column (90 × 15 mm) in Buffer B. The void volume fraction was collected for each sample and assayed for
PA-dependent protein kinase activity using the
phosphocellulose binding assay. The ammonium sulfate-free samples were
also concentrated by centrifugation with Centricon-30 tubes (Amicon;
Beverley, MA) and stored at 70 °C.
Hydrophobic Interaction Chromatography
The 40% ammonium sulfate protein precipitate obtained from 25 mg of cytosolic protein (containing 85% of recovered activity) was resuspended on ice in Buffer B containing 20% ammonium sulfate. After 10 min, the solution was cleared by centrifugation and applied to a Ranin Hydropore column (Woburn, MA), equilibrated with Buffer B containing 10 µg/ml leupeptin, 1 µg/ml pepstatin A, and 20% ammonium sulfate (Buffer C). The unretained proteins were collected, and the retained proteins were then eluted with Buffer C containing no ammonium sulfate. Fractions containing the retained and unretained proteins were concentrated via centrifugation using Centricon 30 tubes and were assayed for PA-dependent protein kinase activity, using the phosphocellulose binding assay, and for the presence of various protein kinases by Western blot analysis.
Gel Filtration Chromatography
Neutrophil cytosol (~120 mg of protein) was adjusted with a solution of saturated ammonium sulfate at 4 °C to yield 40% ammonium sulfate. This mixture was kept on ice for 10 min and was centrifuged (10,000 × g, 4 °C, 10 min) to pellet precipitated protein. The precipitate was resuspended in Buffer B containing 20% ammonium sulfate. After 10 min on ice, the solution was cleared by centrifugation and applied to a Pharmacia Sephacryl S-200 column equilibrated with Buffer C containing no ammonium sulfate. Fractions (4.2 ml) were collected and assayed for PA-dependent protein kinase activity by the phosphocellulose assay. In a separate experiment, neutrophil cytosol (~240 mg of protein) was subjected to 40% saturated ammonium sulfate precipitation. The resulting precipitate was resuspended in Buffer C, and the solution was cleared by centrifugation. The cleared supernatant was then subjected to hydrophobic interaction chromatography as described above. The bound proteins were eluted, concentrated by centrifugation with Centricon-10 tubes, and then applied to the Sephacryl S-200 column, as described above. Fractions were assayed for activity using the SDS-PAGE phosphorylation system. Molecular mass standards (thyroglobulin = void, B-amylase = 200 kDa, alcohol dehydrogenase = 150 kDa, bovine serum albumin = 66 kDa, carbonic anhydrase = 29 kDa, and cytochrome C = 12.4 kDa) were loaded and eluted separately for calibration of the column.
Phosphoamino Acid Analysis
GST-p47-phox was phosphorylated in the presence of
[-32P]ATP, separated by SDS-PAGE as described above,
and transferred to polyvinylidene difluoride (50). The polyvinylidene
difluoride membrane was then washed with H2O and subjected
to autoradiography, and the phosphorylated GST-p47-phox band
was excised from the membrane. The polyvinylidene difluoride membrane
was then subjected to hydrolysis with 5.7 N HCl, at
110 °C for 1 h as described previously (54). Hydrolysis was
stopped by the addition of 200 µl of dH2O, and samples
were centrifuged. The supernatant was then lyophilized, suspended in
phosphoamino acid thin layer chromatography analysis buffer (62.5%
isobutyric acid, 1.9% n-butyl alcohol, 4.8% pyridine, 2.9% glacial acetic acid) containing 1 mg/ml phosphoamino acid standards (55), and spotted on cellulose plates. The plates were then
subjected to thin layer chromatography using the phosphoamino acid
buffer (55). The plates were allowed to air dry, standards were
visualized with ninhydrin (55), and radiolabeled phosphoamino acids were detected by autoradiography.
Immunodepletion of Raf-1 or B-Raf from Neutrophil Cytosol
Cytosol (250 µl) was diluted to 1 ml with 50 mM Tris (pH 7.0), 150 mM NaCl, 2 mM EDTA, and 1 mM EGTA, and incubated with 100 µl of Protein A-Sepharose beads for 10 min, followed by removal of the beads. 10 µg/ml of either anti-Raf-1 antibody, anti B-Raf antibody, or control IgG was added to 330 µl of diluted, precleared cytosol, and the mixture was incubated at 4 °C for 1 h. Protein A-Sepharose beads (100 µl) were added again, and each mixture was incubated, with shaking, at 4 °C for 1 h. The beads were pelleted by centrifugation, and the supernatants were collected and assayed for PA-dependent protein kinase activity by the phosphocellulose binding assay. Immunoprecipitates and the resulting supernatants were analyzed by SDS-PAGE, followed by Western blotting with antibodies to Raf-1 or B-Raf.
Western Blot Analysis
Samples were prepared and separated by 9% SDS-PAGE (48) and
transferred to nitrocellulose (50). The nitrocellulose blots were
blocked by incubation for 1 h in 5% nonfat milk in TBS-T (10 mM Tris, 100 mM NaCl, and 0.1% Tween 20).
Blots were then incubated for 2 h with the appropriate primary
antibody at the following dilutions/concentrations: anti-PKC- and
anti-PKC-
at 2 µg/ml; anti-PKC-
at 1:100;
anti-PKC-
at 1:2000, anti-MAPK at 1:1000, anti-PAK-1 and anti-PAK-2
at 1:1000, anti-phosphotyrosine (4G10) at 1 ng/ml, anti-Raf-1 at 1:1000
and anti-B-Raf at 1:500. Blots were subjected to six 5-min washes. The
blots were then incubated for 1 h with the appropriate horseradish
peroxidase-conjugated secondary antibody at a 1:5000 dilution and
washed again with TBS-T. Blots were visualized by enhanced
chemiluminescence according to manufacturer's recommendations.
Preparation of Opsonized Zymosan
Zymosan was suspended in 154 mM NaCl, boiled for 15 min, centrifuged (10 min, 300 × g, 4 °C), washed once, and resuspended in phosphate-buffered saline. Two volumes of pooled human serum, pooled from four or five healthy donors, and one volume of zymosan suspension were incubated for 30 min at 37 °C (56). The opsonized zymosan was then centrifuged, washed once in stimulation buffer, and resuspended in stimulation buffer for use.
We
have shown previously that the addition of PA to a mixture of cytosolic
and membrane fractions from neutrophils results in the phosphorylation
of a wide range of neutrophil proteins, including p47-phox
(8). We investigated the ability of other phospholipids to induce
protein phosphorylation in this system. Results are shown in Fig.
1. PC, PE, or cardiolipin (CL) did not induce protein
phosphorylation above basal levels (H2O). Since CL was
reported to activate a PA-sensitive protein kinase (19), we also
examined a range of concentrations of this lipid (3 µM to
1 mM). No significant phosphorylation occurred at any of
the CL concentrations tested (data not shown). PS and
phosphatidylinositol each induced a moderate level of protein
phosphorylation. While phosphatidylglycerol induced protein
phosphorylation to a higher degree than PS or phosphatidylinositol,
the best activator of protein phosphorylation was PA. Indeed, the
addition of PA resulted in several proteins being phosphorylated that
were not phosphorylated in the presence of the other phospholipids
(Fig. 1, arrows). This suggests that PA activates a protein
kinase(s) not responsive to other phospholipids.
We next compared the phosphorylation pattern of cytosol stimulated with
PA in vitro with cytosol isolated from opsonized
zymosan-stimulated intact cells. Opsonized zymosan stimulation has been
shown to induce activation of PLD, and this activation has been linked to the activation of NADPH oxidase (46, 57). Increases in protein
phosphorylation were harder to detect in the in vivo system due to high levels of labeled phosphate incorporated in nonstimulated cells. However, increased protein phosphorylation in stimulated samples
could be observed upon longer autoradiographic exposures (Fig.
2). This indicates that a protein kinase is probably
activated by stimulation of neutrophils with opsonized zymosan. Several proteins appeared to be phosphorylated in both the intact cell and the
in vitro system (Fig. 2, arrows). In both
systems, p47-phox was phosphorylated upon stimulation. This
suggests that the protein kinase activated by PA in the in
vitro system is also activated in the intact cell, under
conditions that result in PLD activation. Taken together, these data
indicate that PA is the most effective activator of protein
phosphorylation in the neutrophil cell-free system and suggest that the
protein kinase activated in the cell-free system is also activated
under physiological conditions.
Subcellular Location of the PA-activated Protein Kinase
To
determine the subcellular location of the protein kinase activated by
PA, we used recombinant p47-phox (Rp47) as an exogenous substrate for the enzyme. Rp47 was added in excess to phosphorylation reaction mixtures that contained either cytosol and membranes, cytosol
alone, or membranes alone. Phosphorylation of Rp47 was dependent upon
the presence of PA (Fig. 3). Reaction mixtures that
lacked cytosol did not result in PA-dependent
phosphorylation of Rp47. In contrast, reaction mixtures that lacked the
membrane fraction supported PA-dependent phosphorylation.
Shown in Fig. 3 is a 5-min autoradiograph. In lanes with cytosol
present, protein phosphorylation patterns similar to those seen in Fig.
1 (PA lane) were observed when longer exposures were viewed.
Minimal protein phosphorylation was observed in longer exposures when
reaction mixtures contained only membrane fractions (data not shown).
The identical pattern was observed when the GST-p47-phox
fusion protein was used as the exogenous substrate (data not shown).
These data indicate that the PA-activated protein kinase is cytosolic
and does not require membrane components for activation.
Comparison of Protein Kinase Activation by PA and cis-Unsaturated Fatty Acids
Rat liver (19, 20) and platelets (18) contain
cytosolic PA-responsive protein kinases, which were identified as
cardiolipin-activated and cis-unsaturated fatty
acid-activated protein kinases, respectively. Recently, the hepatocyte
protein kinase has been determined to be a novel protein kinase (PKN)
(16), which is also the PKC-related protein kinase 1 (PRK1) (14, 17,
58). We tested the possibility that the protein kinase activity we
observe in the neutrophil is the same or similar to these PA-responsive
protein kinases. The results shown in Fig. 1 suggest the neutrophil
enzyme is not a cardiolipin-activated protein kinase. We tested whether
the cis-unsaturated fatty acid AA could activate a protein
kinase in neutrophil cytosol. Fig. 4 shows protein
kinase activation as a function of PA and AA concentration, using the
phosphocellulose assay. AA was a poor inducer of protein kinase
activity with maximal activity of 40 ± 3 pmol of
PO4/min/mg (25 µM AA, Fig. 4). In contrast, PA activated a protein kinase(s) in a dose-dependent manner
with a 6-fold higher maximal activity (246 ± 44 pmol of
PO4/min/mg, 30 µM PA, Fig. 4). Another
cis-unsaturated fatty acid, oleic acid, also elicited little
protein kinase activation (35 pmol of PO4/min/mg, 25 µM oleic acid, n = 2, data not shown).
Thus, the PA-responsive protein kinase found in neutrophil cytosol is
poorly activated by cis-unsaturated fatty acids or
cardiolipin and does not appear to be a fatty acid-activated protein
kinase described by others (14, 16-20).
Separation of the PA-activated Protein Kinase from PKC Isoforms
PA has been shown, in vitro, to activate
several isoforms of PKC (9-13), raising the possibility that the
protein kinase target of PA in neutrophil cytosol is one or more of
these enzymes. To test this possibility, we determined whether the PKC
isoforms present in neutrophil cytosol (59-64) co-purified with the
PA-activated protein kinase. We first subjected neutrophil cytosol to
sequential ammonium sulfate precipitation, as described under
"Experimental Procedures." The fractions obtained were tested for
the location of the PA-activated protein kinase, using the
phosphocellulose protein kinase assay. The PA-activated protein kinase
was recovered primarily in the 40% ammonium sulfate precipitate, which
contained 85% of the total recovered PA-activated protein kinase
activity (Fig. 5A). Much less activity was
observed in the 70% precipitate and the resulting supernatant (13 and
2% of the total recovered activity, respectively). The same fractions
were then evaluated for the presence of various PKC isoforms by Western
blotting (Fig. 5B). The PKC isoforms ,
,
, and
were present in unfractionated cytosol
that had been passed over a Sephadex G-25 column as a control for the
ammonium sulfate-treated fractions (G25). Following ammonium
sulfate fractionation of the cytosol and Sephadex G-25 chromatography of the fractions, the
calcium-dependent PKC isoforms,
and
, were recovered in the 70% ammonium sulfate
precipitate. Similarly, the calcium-independent isoforms,
and
,
were recovered predominantly in the 70% ammonium sulfate precipitate,
although small amounts of PKC-
and -
were present in the 40%
ammonium sulfate precipitate.
Since PKC- and -
remained in the 40% ammonium sulfate
precipitate, we subjected the PA-activated protein kinase to an
additional step of purification. The 40% ammonium sulfate pellet was
resuspended in buffer containing 20% ammonium sulfate and subjected to
hydrophobic interaction chromatography using a Rainin Hydropore column.
The unretained proteins (Void) and the retained proteins
(Bound) were collected as described under "Experimental
Procedures." Activity measurements (Fig.
6A) showed that the PA-activated protein
kinase was predominantly present in the retained protein fraction
(Bound, 75% of the recovered PA-activated protein kinase
activity). The unretained protein and the retained protein fractions
were then subjected to Western blot analysis for the presence of
PKC-
and -
. Results (Fig. 6B) show that neither
PKC-
nor -
were retained on the column; these PKC isoforms were
observed solely in the unretained protein fraction (Void).
The retained protein fraction (Bound) was also analyzed, by
Western blotting, for the presence of PKC isoforms not known to be
present in the neutrophil (
,
,
,
), to determine if one of
these isoforms was present. No immunoreactive bands were observed (data
not shown). These data indicate that the PA-activated protein kinase is
not likely to be a PKC isoform.
Separation of the PA-activated Protein Kinase from PAK and MAPK Isoforms
Neutrophil cytosol contains MAPK and PAK isoforms, two
classes of protein kinases recently shown to phosphorylate
p47-phox, the in vitro substrate for the
PA-activated protein kinase (24, 25, 29, 31). While neither of these
protein kinases are known to be directly activated by PA, it was
possible that these protein kinases are targets for PA in our system or
are activated downstream of the PA-activated protein kinase. Neutrophil
cytosol depleted of Raf-1 or B-Raf by nondenaturing immunoprecipitation still maintained PA-dependent protein kinase activity (data
not shown), suggesting that neither Raf-1 nor B-Raf is the target for
PA in this system. This also suggests that MAPK isoforms are not
activated via PA-mediated activation of Raf-1. However, it was possible
that MAPK isoforms may be activated directly by PA. Fractions obtained
by ammonium sulfate precipitation and hydrophobic interaction
chromatography, during the partial purification of the PA-activated
protein kinase, were analyzed for the presence of MAPK or PAK by
Western blotting. As presented in Figs. 5A and 6A, the PA-activated protein kinase was precipitated from a
40% saturated ammonium sulfate solution (40% cut = 85% of recovered activity) and was retained by the column resin during
hydrophobic interaction chromatography (Bound = 75% of
recovered activity). Fig. 7 shows that both PAK-2 and
p42 and p44 MAPK isoforms are present in neutrophil cytosol. Upon 40%
saturated ammonium sulfate precipitation, both kinase families
distributed between the 40% cut and the resulting supernatant,
although MAPK was predominantly in the 40% cut. In contrast to the
PA-dependent protein kinase, both PAK and MAPK isoforms
were found only in the unretained protein fraction eluting from the
hydropore column. Similar results to those obtained with PAK-2
antibodies were obtained with antibodies recognizing PAK-1 (data not
shown). Thus, the PA-activated protein kinase co-purifies with neither
PAK-1 or PAK-2 nor p42 or p44 MAPK isoforms.
Molecular Weight Determination of the PA-activated Protein Kinase
The apparent molecular weight of the
PA-dependent protein kinase was determined by gel
filtration chromatography. Neutrophil cytosol was first subjected to
ammonium sulfate precipitation and then gel filtration chromatography,
as described under "Experimental Procedures." The peak of
PA-dependent protein kinase activity eluted at fraction 28 (Fig. 8A), which corresponds to a molecular mass of 125 kDa. We confirmed these results with more purified material
obtained by subjecting neutrophil cytosol to both ammonium sulfate
precipitation and hydrophobic interaction chromatography prior to gel
filtration chromatography. Fractions were analyzed by the SDS-PAGE
phosphorylation assay. Fig. 8B shows that, like the activity
seen in Fig. 8A, the peak of GST-p47-phox
phosphorylation occurred at fraction 28. These data indicate that the
PA-responsive protein kinase has a maximum size of 125 kDa.
Characterization of the PA-activated Protein Kinase
Due to
the low yield (4%) after gel filtration, we used the hydropore-bound
material to characterize the PA-activated protein kinase. We first
analyzed the effect of calcium and GTPS on the activation of the
PA-activated protein kinase. We found that calcium slightly enhanced
the PA-activated protein kinase (1571 pmol of PO4/min/mg in
the absence of calcium, 1780 pmol PO4/min/mg in the
presence of 0.6 mM calcium, n = 2). The
addition of 1 µM GTP
S, sufficient to activate Rac in
the NADPH oxidase system (7, 8, 65, 66), had little effect on
activation (1680 pmol of PO4/min/mg activity in the absence
of GTP
S, 1613 pmol PO4/min/mg in the presence of
GTP
S, n = 2). We also verified that the partially purified PA-activated protein kinase phosphorylated recombinant p47-phox (Rp47), using SDS-PAGE and autoradiography (data
not shown). The PA-activated protein kinase was also active in the presence of mixed vesicles, containing PC, PS, and PE, which mimicked the inner membrane lipid layer (67). The concentration of PA yielding
maximal protein kinase activation shifted, only slightly, from 30 µM PA (3830 pmol PO4/min/mg,
n = 2) in the absence of mixed vesicles to 100 µM PA (3634 pmol PO4/min/mg,
n = 2) in the presence of mixed vesicles. This suggests
that activation of the PA-activated protein kinase can occur in a
physiologically relevant manner.
We have previously shown that NADPH oxidase activation and endogenous
protein phosphorylation induced by PA plus DG is sensitive to several
protein kinase inhibitors (8). Fig. 9 shows that the
partially purified PA-activated protein kinase is sensitive to the same
inhibitors. In the absence of inhibitors, the GST-p47-phox fusion protein was phosphorylated by the protein kinase only when PA
was present. Staurosporine and C-I each completely inhibited the
phosphorylation of GST-p47-phox, while GF-109203X was less effective. The autoradiograph shown in Fig. 9 is a long exposure to
show that little protein phosphorylation occurred in the presence of
these protein kinase inhibitors. Staurosporine is known to inhibit both
protein serine/threonine kinases and protein-tyrosine kinases (68).
C-I, an H-7 analog, inhibits serine/threonine protein kinases but not
protein-tyrosine kinases (51). This suggests that the PA-activated
protein kinase is a protein serine/threonine kinase.
To further evaluate the amino acid specificity of the protein kinase,
we performed phosphoamino acid analysis on phosphorylated GST-p47-phox. Fig. 10A clearly
shows that serine residues are phosphorylated and that threonine
residues are not. However, this analysis did not clearly separate
phosphotyrosine from inorganic phosphate. To determine if tyrosine
residues were phosphorylated, we extracted each of the phosphoamino
acids from the cellulose plate with methanol. This treatment did not
elute inorganic phosphate (data not shown). The methanol extracts were
then spotted onto cellulose plates and subjected to autoradiography.
After extraction, only the phosphoserine and phosphotyrosine extracts
resulted in spots, as assessed by autoradiography (data not shown).
This confirmed that serine residues were phosphorylated and indicated
that tyrosine residues were also phosphorylated. We confirmed tyrosine
phosphorylation by Western blot analysis. The GST-p47-phox
fusion protein became phosphorylated on tyrosine residues only when PA
was present (Fig. 10B). It was possible that the GST portion
of the fusion protein was phosphorylated by a protein kinase in the
hydropore-bound material. However, we found that hydropore-bound
material did not phosphorylate the cleaved GST portion of the fusion
protein in the presence of PA (data not shown). In addition,
recombinant p47-phox was phosphorylated on tyrosine
residues, in a PA-dependent manner, using cytosol as a
source for protein kinases (data not shown). Taken together, these data
suggest that the hydropore-bound material contains one or more protein
kinases that phosphorylate serine and tyrosine residues of
p47-phox in a PA-dependent manner. Since no
mammalian dual specificity protein kinases that phosphorylate both
serine and tyrosine residues have been identified, we speculate that
the hydropore-bound material contains at least two PA-responsive protein kinases.
Cell and Tissue Distribution of PA-activated Protein Kinase
Since PA is generated in many cell types upon stimulation of PLD (2), we investigated whether cytosol from other cell types contained a protein kinase responsive to PA. Cytosols were isolated as described under "Experimental Procedures," and protein kinase activity was determined in the presence and absence of PA. Several cell types were analyzed. PA-dependent protein kinase activity (defined here as >2-fold stimulation upon PA addition) was present in all except three cell lines (HepG2, McA-RH7777, and IMR-32). The highest levels of PA-dependent protein kinase activity were present in neutrophil (305 ± 57 pmol of PO4/min/mg, n = 6), lymphocyte (492 ± 55 pmol of PO4/min/mg, n = 4), HL-60 (598 pmol of PO4/min/mg, n = 2), THP-1 (346 ± 92 pmol of PO4/min/mg, n = 3), and rat brain (528 ± 66 pmol of PO4/min/mg, n = 4) cytosols. Platelet (132 ± 53 pmol of PO4/min/mg, n = 5), CFTL-15 (153 pmol of PO4/min/mg, n = 2), RAW 267.4 (241 pmol of PO4/min/mg, n = 2), K562 (130 pmol of PO4/min/mg, n = 1), and Madin-Darby canine kidney cell (104 ± 46 pmol of PO4/min/mg, n = 3) cytosols had intermediate levels of activity. Thus, PA-responsive protein kinases exist in cells other than neutrophils.
We further investigated the possibility that the same enzyme present in neutrophil cytosol might be present in rat brain cytosol. Rat brain cytosol was subjected to 40% saturated ammonium sulfate precipitation followed by hydrophobic interaction chromatography. The purification profile obtained was similar to that obtained with neutrophil cytosol (see Figs. 4 and 5). A PA-dependent protein kinase was precipitated by 40% saturated ammonium sulfate (980 pmol of PO4/min/mg, n = 1) and was retained during hydrophobic interaction chromatography (1590 pmol of PO4/min/mg, n = 1). This suggests that the same or a similar enzyme was present in these two sources of material. However, other PA-responsive protein kinases appear to be more prevalent in the rat brain cytosol than in neutrophil cytosol, since the supernatant from the 40% ammonium sulfate cut contained 57% of the recovered activity, compared with 15% using neutrophil cytosol. This may account for the smaller increase in specific activity observed in rat brain material (1.8-fold increase) compared with neutrophil material (5-fold increase) after elution from the Hydropore column.
We have recently developed a phosphorylation-dependent cell-free system for the activation of the neutrophil NADPH oxidase (7, 8). In this system, PA activates one or more protein kinases that phosphorylate the NADPH oxidase component p47-phox. Here, we have eliminated known protein kinase targets for PA in this system. Our results suggest that PA activates potentially two previously unidentified cytosolic protein kinases, which phosphorylate p47-phox.
We first characterized the lipid specificity for the activation of the PA-dependent protein kinase. Previous studies indicated that DG has only minor enhancing effects on protein phosphorylation induced by PA (8), suggesting that DG-regulated PKC isoforms were unlikely to be targets for PA in this system. Fig. 1 demonstrates that PA induced a high level of protein phosphorylation and activates a protein kinase(s) that is not a target for other phospholipids, supporting the concept that PA activates a novel protein kinase. Bocckino et al. (69) first demonstrated that PA could induce protein phosphorylation in various rat tissues, and our results confirm this. Like them, we found that PC and PE induced minimal protein phosphorylation and that phosphatidylinositol and PS induce moderate levels (Fig. 1). We show here that phosphatidylglycerol can also induce protein phosphorylation (Fig. 1). In the neutrophil system, more proteins appear to be substrates for a PA-dependent protein kinase(s) than in rat tissues.
We considered the possibility that the protein kinase target for PA was
one of the recently described fatty acid-activated protein kinases
(18-20), one of which has been shown to be PKN (16), known to be
activated by cis-unsaturated fatty acids as well as CL (17)
and identical to PRK1 (14, 17). The predicted molecular size of
PRK1/PKN is 120 kDa (14, 15, 17, 58, 70), similar to the apparent size
of the predominant protein kinase described here (Fig. 8). PA was much
more effective than cis-unsaturated fatty acids at inducing
protein kinase activity (Fig. 4 and text). In addition, CL (3 µM to 1.0 mM), was unable to induce protein
phosphorylation in neutrophil cytosol (Fig. 1 and text). Furthermore,
while PKN is regulated by the GTPase Rho (71, 72), the
PA-dependent protein kinase activity described here was
independent of GTPS. Thus, it is unlikely that Rho could activate
PKN under these conditions. These data strongly suggest that PRK1/PKN
(14, 15, 17, 58, 70) and/or the fatty acid-activated protein kinases
previously described (18-20) are not responsible for the
PA-dependent protein kinase activity in neutrophils. It is
possible that the neutrophil PA-activated protein kinase is related to
these enzymes and is an isoform poorly responsive to
cis-unsaturated fatty acids and highly responsive to PA.
Such a determination will require identification of the neutrophil enzyme at the molecular level.
Our second approach was to purify the PA-activated protein kinase present in neutrophil cytosol to a stage where we could distinguish the PA-activated protein kinase from other protein kinase targets. The partial purification protocol successfully separated the PA-activated protein kinase(s) from a number of other protein kinases in neutrophils known to phosphorylate p47-phox, including PKC, MAPK, and PAK isoforms (Figs. 5, 6, and 7). We did not examine fractions for the presence of PKC-µ, which has not been reported to be in neutrophils. Since PKC-µ has a predicted molecular size of 115 kDa (73), it is possible that this isoform is responsible for some of the PA-dependent protein kinase activity in neutrophils. However, PKC-µ has been reported to have a putative transmembrane domain (73) and has been isolated in the particulate material after subcellular localization (74). In contrast, the PA-activated protein kinase is cytosolic. Also, PA has been reported to have little effect on activation of PKC-µ (75). Taken together, these observations strongly suggest that PKC-µ is not a PA-activated protein kinase in neutrophil cytosol.
MAPK and PAK isoforms were also candidates for the protein kinase(s)
responsible for phosphorylation of p47-phox (24, 25). However, neither p42 or p44 MAPK isoforms nor PAK-1 or PAK-2 isoforms co-purified with the PA-activated protein kinase (Fig. 7 and text). In
addition, depletion of cytosolic Raf-1 or B-Raf, which could lead to
MAPK activation, by immunoprecipitation had little or no effect on
PA-dependent protein kinase activity (see text). Finally,
the PA-dependent protein kinase activity was
GTPS-independent, which suggests Rac was not participating in
protein kinase activation in our system. We did not examine other
protein kinases in these families (24, 28-31, 76).
These results strongly suggest that PA activates a novel protein kinase in neutrophil cytosol, which phosphorylates the NADPH oxidase component p47-phox. The PA-activated protein kinase was sensitive to the same protein kinase inhibitors that inhibited cell-free NADPH oxidase activation by PA plus DG (Fig. 9) (8). GF-109203X has been described as a selective PKC inhibitor (77), but it was partially effective against the hydropore-purified PA-activated protein kinase. This may indicate that a PA-activated protein kinase is similar to PKC; however, this remains to be determined. The PA-activated protein kinase phosphorylates p47-phox on serine residues (Fig. 10A), which has been shown to occur in vivo (25, 78, 79). The inhibitor results suggested strongly that the PA-activated enzyme is a protein serine/threonine kinase. However, the phosphoamino acid analysis revealed that PA also activates a protein tyrosine kinase, which phosphorylates p47-phox (Fig. 10, A and B). Thus, the hydropore-bound material either contains at least two PA-responsive protein kinases, or one of the kinases is activated downstream of the other. It is possible that the known lability of the phosphotyrosine bond (54, 55) accounts for the apparent serine/threonine specificity observed during SDS-PAGE analysis. This also may explain the appearance of only one peak of PA-dependent protein kinase activity during gel filtration (Fig. 8). Further purification of these enzymes will be necessary to address these issues and is beyond the scope of this paper.
Tyrosine phosphorylation of p47-phox has not been reported until now. Previous studies have analyzed the phosphorylation of p47-phox after stimulation of neutrophils with phorbol myristate acetate (25, 78, 79). Further work needs to be done to examine if other NADPH oxidase agonists stimulate tyrosine phosphorylation of p47-phox, in vivo. In these studies, it will be important to monitor the hydrolysis of p47-phox to maximize the ability to observe potential tyrosine phosphorylation, since phosphotyrosine is more labile than phosphoserine and phosphothreonine (54, 55). It is also likely that the phosphorylation of p47-phox on tyrosine residues occurs to a lesser extent than serine phosphorylation. Thus, the amount of p47-phox recovered prior to phosphoamino acid analysis and the assay conditions used are likely to be critical to observe tyrosine phosphorylation in vivo. P47-phox contains several tyrosine residues that may be a target for a protein-tyrosine kinase (80-82). Further work is necessary to determine which tyrosine residues are phosphorylated in response to PA. Once these sites are determined, the role of such phosphorylation can be examined more closely. It remains to be determined what role, if any, tyrosine phosphorylation of p47-phox has in NADPH oxidase activation.
A primary significance of this study lies in the possibility that the PA-activated protein kinases are new and selective targets for intracellular PA generated by PLD activation. In neutrophils, the PA-activated protein kinase(s) could participate in the regulation of a variety of cell functions (1, 2, 83), such as activation of the NADPH oxidase. Activation of PLD and the resulting production of PA have been closely linked to the activation of the oxidase (4-8, 84). Our previous data clearly indicate that a PA-activated protein kinase participates in the PA- and DG-dependent cell-free activation of NADPH oxidase (8). A PA-activated protein kinase retained the ability to phosphorylate p47-phox, even after three steps of purification (Fig. 8B), providing additional evidence indicating that the NADPH oxidase component p47-phox is a substrate for the enzyme. However, it remains to be determined if phosphorylation of p47-phox by a PA-activated protein kinase regulates the assembly and activation of the NADPH oxidase enzyme.
It is notable that we found PA-dependent protein kinase activity in a wide range of blood cells and hematopoietic cell lines and in rat brain cytosol, suggesting that the enzyme may be widely distributed. Activity was not apparent in the hepatoma and neuroblastoma cell lines, but the significance of these preliminary observations is not known. It is not yet clear if the same enzymes are responsible for the activity in all of these cells and in rat brain. However, like the enzyme in neutrophils, a PA-activated protein kinase in rat brain cytosol was precipitated at 40% ammonium sulfate saturation and retained on the Hydropore column. Once a PA-activated protein kinase has been purified and sequence information is available, it will be possible to use molecular and immunological approaches to definitively address the cell and tissue distribution of the enzyme.
In conclusion, we have shown that neutrophil cytosol contains potentially two novel PA-responsive protein kinases. The molecular weight of one of these protein kinases is 125 kDa. We have differentiated these enzymes from other known protein kinases that could be targeted by PA in our system and have evidence that the enzymes may be widely distributed in cells and tissues. These protein kinases phosphorylate p47-phox, a necessary component of the NADPH oxidase. Future work will focus on purifying and sequencing the PA-responsive protein kinases to identify them as either novel enzymes or known protein kinases with a new function. In addition, we plan to define the role of PA-activated protein kinases in the phosphorylation-dependent activation of the NADPH oxidase.
We thank the following people at Wake Forest University Medical Center: Dr. Susan Hutson for the use of fast protein liquid chromatography equipment; Drs. B. Moseley Waite, Larry Daniel, Mary Beth Fasano, Charles McCall, Greg Shellness, and Osvaldo Delbono for cell lines; Drs. Carol Cunningham and Susan Hutson for rat brain tissue; Mary Ellenburg for help with Western blot analyses, and Ross Waite for graphics. We also thank Drs. Arie Abo and Gary Bokoch for antibodies to PAK. We are grateful to Dr. Tom Leto for purified recombinant p47-phox and E. coli containing cDNA for GST-p47-phox and to Drs. Sujoy Ghosh and Jay Strum for sharing results prior to publication.