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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 1121-1130
Regulation of p21rac Activation in Human Neutrophils
By
Niels Geijsen,
Sanne van Delft,
Jan A.M. Raaijmakers,
Jan-Willem
J. Lammers,
John G. Collard,
Leo Koenderman, and
Paul J. Coffer
From the Department of Pulmonary Diseases, University Hospital
Utrecht, Utrecht; and The Netherlands Cancer Institute, Division of
Cell Biology, Amsterdam, The Netherlands.
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ABSTRACT |
The small guanosine triphosphate (GTPase) p21rac is highly expressed
in human neutrophils where it is thought to play a role in cytoskeletal
reorganization and superoxide production. Using the p21rac binding
domain of PAK (PAK-RBD) as an activation-specific probe, we have
investigated agonist-stimulated activation of p21rac. Stimulation of
neutrophils with the chemoattractants fMet-Leu-Phe (fMLP) or
platelet-activating factor (PAF) induced an extremely rapid and
transient p21rac activation, being optimal within 5 seconds. This
activation correlates with the rapid changes of intracellular free
Ca2+ ([Ca2+]i) stimulated by
fMLP; however, changes in [Ca2+]i were
neither sufficient nor required for p21rac activation. Furthermore,
fMLP-induced p21rac activation was not inhibited by broad tyrosine
kinase inhibitors or specific inhibitors of ERK, p38 mitogen activated
protein kinase, Src, or phosphatidylinositol 3-kinases. Surprisingly,
the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF)
and tumor necrosis factor- did not cause p21rac activation or
modulate fMLP-induced p21rac activation. AlF , a potent
activator of heterotrimeric G-protein -subunits, however, was found
to activate p21rac. Stimulation of neutrophils with phorbol myristate
acetate (PMA) strongly activated the respiratory burst,
but did not induce p21rac activation, suggesting that superoxide production per se can occur independently of p21rac activation. These
data suggest that in human granulocytes, G-protein coupled receptors,
but not cytokine receptors, activate p21rac via a rapid, novel
exchange-mechanism independently of changes in
[Ca2+]i, tyrosine phosphorylation, or PI3K.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
NEUTROPHILS PLAY an important role in the
host defense to microbial pathogens. Stimulation of these cells induces
multiple responses, including cell adhesion, migration, secretion,
phagocytosis, and the generation of reactive oxygen
species.1 Therefore, neutrophil function is under tight
control, and deregulated activation of neutrophils is implicated in the
pathogenesis of a variety of inflammatory diseases, which are
characterized by tissue damage.2-5 A diverse array of
receptors are expressed on the surface of neutrophils, allowing
regulation by a wide range of agonists and second
messengers.1 Tyrosine kinase-linked receptors such as the
granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, and
serpentine receptors including the N-formylmethionylleucylphenylalanine
(fMLP) receptor activate both distinct and overlapping signaling
pathways regulating neutrophil responses.6-14 Several small
guanosine triphosphatases (GTPases) have been implicated in controlling
neutrophil function. Activation of the p21ras15,16 and
p21rap17 by cytokines and chemoattractants has been
reported in human neutrophils, although their precise roles in
neutrophil functioning are unknown.
Another small GTPase that has been implicated in neutrophil functioning
is p21rac.18-20 p21rac has been shown to be present in the
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex
where it is thought to play a critical role in the production of oxygen
radicals.18,21-26 In several cell lines, activation of
p21rac has been found to be essential for superoxide
production.24,27
However, measurement of the activation of p21rac in cells has been
hampered by the lack of an immunoprecipitating antibody. GTP-loading of
endothelial cells transfected with epitope-tagged p21rac has, however,
been reported and shown to be blocked using specific inhibitors of
phosphatidylinositol-3 kinase (PI3K), suggesting that p21rac is
downstream of this lipid kinase.28 Like other small
GTPases, p21rac activity is controlled by switching from its inactive,
guanosine diphosphate (GDP) bound state to the active, GTP bound state.
Guanine nucleotide exchange factors (GEFs) activate Rac by facilitating
the exchange of GDP for GTP. Thus far, two GEFs for p21rac have been
extensively studied: Vav and Tiam1.29,30 Vav is expressed
predominantly in hematopoietic cells and its tyrosine phosphorylation
is promoted by binding to PI3K-phosphorylated lipids.31 In
these cells, Vav has been shown to be activated by stimuli that
activate PI3K, including GM-CSF.32
While much work has evaluated the role of p21rac in cellular
functioning by overexpression of dominant-negative or dominant-active mutants in cell lines, as mentioned above, it has not been possible to
measure activation of p21rac by cellular stimuli. Here we use a novel
assay to measure the p21rac activation.33 This assay uses
the p21rac binding domain of PAK (PAKcrib), which has a high affinity
for p21racGTP, but no affinity for p21racGDP, to precipitate activated
p21rac from cell lysates.34-36 We show for the
first time that in human neutrophils, activators of G-protein coupled receptors (GPCRs), but not cytokine receptors, activate p21rac. The
activation of p21rac by fMLP is independent of the elevation of
[Ca2+]i, activation of tyrosine kinases and
PI3K and does not correlate with the activation of the respiratory
burst. This suggests a novel pathway for activation of exchange factors
regulating p21rac in these cells.
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MATERIALS AND METHODS |
Isolation of human neutrophils.
Blood was obtained from healthy volunteers from the Red Cross Blood
Bank (Utrecht, The Netherlands). Mixed granulocytes were isolated from
the buffy-coat of 500 mL 0.4% (wt/vol) trisodium citrate (pH
7.4)-treated blood as previously described.37 Mononuclear cells were removed by centrifugation over isotonic Percoll (1.078 g/mL)
from Pharmacia (Uppsala, Sweden). After lysis of the erythrocytes in
isotonic NaCl solution, neutrophils were washed and resuspended in
incubation buffer (20 mmol/L HEPES, 132 mmol/L NaCl, 6 mmol/L KCI, 1 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 5 mmol/L glucose, 1 mmol/L CaCI2) containing 0.5% human
serum albumin (HSA; Central Laboratory of The Netherlands Red Cross
Blood Transfusion Service, Amsterdam). Neutrophils were incubated for
30 minutes at 37°C before stimulation. In all experiments, a
concentration of 106 cells/mL was used for stimulation.
Figures are representative of at least three other experiments from
independent donors.
Neutrophil stimulation.
One milliliter of neutrophil suspension was stimulated with one of the
following stimuli: fMLP (1 µmol/L to 10 nmol/L), platelet-activating factor (PAF) (1 µmol/L), tumor necrosis factor- (TNF-) (100 U/mL), phorbol myristate acetate (PMA) (100 ng/mL), thapsigargin (100 nmol/L) (all from Sigma, St Louis, MO), GM-CSF (1010
mol/L; Genzyme, Boston, MA), and ionomycin (100 nmol/L; Calbiochem, La
Jolla, CA). The concentrations used are known to prime neutrophils or
activate the respiratory burst.38,39 In some experiments, cells were preincubated as described in the legends of the figures with
one of the following inhibitors: PP1, PD098059, and LY294002 (all from
Biomol, Plymouth, PA) and SB203580 (Alexis, Laufelfingen, Switzerland).
At different time points, 500 µL 3X Lysis Buffer (see below) was
added, samples were vortexed and lysed on ice for 10 minutes. For the
AlF4 stimulation, neutrophils were
resuspended at 106 cells per 500 µL HEPES3+.
Cells were stimulated by adding 500 µL aluminum fluoride
(AMF) buffer (20 mmol/L HEPES, 132 mmol/L NaCl, 6 mmol/L
KCl, 1 mmol/L MgSO4, 1.2 mmol/L
KH2PO4, 5 mmol/L glucose, 1 mmol/L
CaCl2, 0.5% HSA, 60 mmol/L NaF, 100 µmol/L
AlCl3) and incubated at 37°C. At different time points,
cells were lysed by addition of 3X Lysis Buffer, vortexed, and lysed on
ice for 10 minutes.
Rac-1 activation assay using GST-PAKcrib.
The Rac and CDC42 binding domain of PAK1b (accession no.
AF 071884), crib domain (aa 56-272), was generated and used as
previously described.33 GST pull-down assays were performed
essentially as described for Ras and Rap.14,15 Briefly,
human neutrophils (106 cells/mL) or BW5147 cell
lines40 (107 cells/mL) were lysed for 10 minutes at 4°C by addition of 3X lysis buffer (1X lysis buffer: 50 mmol/L Tris pH 7.4, 10% glycerol, 200 mmol/L NaCl, 1% NP-40, 2 mmol/L
MgCl2, 1 mmol/L phenylmethyl sulfonyl fluoride [PMSF], 1 mmol/L benzamidine, 10 µg/mL aprotinin, and 10 µg/mL leupeptin).
Lysates were cleared by centrifugation and GST-PAKcrib protein was
added for 30 minutes at 4°C and subsequently washed three times in
lysis buffer. The beads were boiled in Laemmli sample buffer and
protein samples were separated by 15% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred
to polyvinylidene difluoride membranes (Immobilon-P; Millipore,
Bedford, MA). The membranes were probed with anti-Rac antibodies (Transduction Laboratories, Lexington, KY) and
sheep anti-mouse peroxidase-conjugated antibodies (DAKO,
Glostrup, Denmark). Immune complexes were detected by
enhanced chemiluminescence (Amersham, Buckinghamshire, UK). The Ras
activation assay using Raf-RBD was performed as previously
described.15,41 All experiments were repeated three times.
Blots are representative of at least three independent experiments
For the in vitro Rac-loadings, p21rac protein was expressed and
purified as previously described.42 A total of 0.2 µg of purified p21rac was added to 1 mL of lysis buffer containing either GDP
or the nonhydrolyzable GTP analogue GppNHp
(5'-guanosyl-[ , -imido]-triphosphate). GST-PAK protein was
added and incubated for 30 minutes to allow binding. After washing
three times, samples were analyzed by SDS-PAGE as described above.
Depletion of intracellular free Ca2+.
Intracellular free Ca2+ was depleted as previously
described.43 Neutrophils were suspended in Ca2+
free incubation buffer supplemented with 1 mmol/L EGTA. Indo-1/AM (Molecular Probes, Eugene, OR) was added to 1-mL aliquots of suspended cells (106 cells/mL) at a final concentration of 1.5 µmol/L. Cells were incubated for 40 minutes at 37°C. Thapsigargin
(100 nmol/L) was added 10 minutes before washing to deplete internal
stores. Cells were washed once with Ca2+ free incubation
buffer containing EGTA. Determination of p21rac activation and
Ca2+ measurements were performed in parallel with the same
cells. Calcium concentration was measured by a dual excitation at a
wavelength of 340 nm and detected at 390 nm using a Hitachi F4500
fluorescence spectrophotometer (Hitachi, Tokyo,
Japan).43
Respiratory burst measurements.
Neutrophils were preincubated in the absence or presence of GM-CSF
(1010 mol/L for 20 minutes). fMLP (1 µmol/L), PMA
(100 ng/mL), or ionomycin (100 nmol/L) was added to the cells and
oxygen consumption was measured using a Clark oxygen electrode (YSI
Inc, Yellow Springs, OH) as previously
described.44 In addition, cells were also pretreated with
or without LY294002 for 20 minutes.
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RESULTS |
GST-PAKcrib can be used as a specific probe for p21rac-GTP.
The responses of human phagocytes to appropriate stimuli can result in
cellular migration or production of superoxide resulting in microbial
killing. Both these processes have been linked to activation of the
small GTPase p21rac.2,18 Previously, it has been impossible
to measure the activation of this GTPase due to the inability to
immunoprecipitate endogenous protein from cell lines or primary
cells.28 However, it has been demonstrated that a
GST-PAKcrib fusion protein specifically binds to GTP-bound, but not
GDP-bound p21rac, with high affinity.33-36 Indeed,
neutrophils stimulated with fMLP demonstrate activation of
PAKs.45,46
To confirm that GST-PAKcrib could be used as a specific probe to
monitor the activation of p21rac, recombinant human p21rac was loaded
with either GDP or the GTP analogue GppNHp. GST pull-down was performed
using GST-PAKcrib, followed by anti-p21rac immunoblotting. As shown in
Fig 1A, GST-PAKcrib strongly associated
with the GTP-loaded p21rac, whereas no association with the GDP-loaded
p21rac was observed (Fig 1A, top panel). On the same blot, Coomassie
staining was performed demonstrating the use of equal amounts of
GST-PAKcrib in both lanes (Fig 1A, middle panel). Before performing the
GST pull-down, 1/40 of each sample was analyzed on Western blot,
showing equal amounts of p21rac were present in both samples (Fig 1A, bottom panel). To determine whether this assay was also applicable for
the analysis of p21rac activity in vivo, BW5147 cells stably retrovirally transduced with control vector (Fig 1B, lane 1) or with
epitope tagged p21rac(V12) (lane 2) were used. GST pull-down was
performed using GST-PAKcrib, followed by anti-p21rac immunoblotting. GST-PAKcrib strongly associated with p21rac(V12), despite its low
expression compared with endogenous p21rac (Fig 1B, bottom panel),
demonstrating that GST-PAKcrib specifically associates with activated,
GTP-bound p21rac. The band running below the epitope-tagged p21rac(V12)
was observed in both lanes due to the association of a small fraction
of activated endogenous p21rac with GST-PAKcrib. To investigate the
specificity of GST-PAKcrib toward p21rac, BW5147 cells stably
retrovirally transduced with control vector (Fig 1C, lane 1), or
epitope-tagged p21rac(V12) (lane 2), p21rho(V14) (lane 3), or
p21ras(V12) (lane 4)40 were used. GST pull-down was
performed using GST-PAKcrib followed by anti-epitope immunoblotting. Although the expression levels of p21rac(V12), p21rho(V14), and p21ras(V12) were equal (Fig 1C, middle panel), only p21rac(V12) was
found to bind GST-PAKcrib. Furthermore, the antibody used for the
detection of p21rac was highly specific, and did not show cross-reactivity with either p21rho or p21ras (Fig 1C, bottom panel).
Because no association of GST-PAKcrib with other active small GTPases
p21rho and p21ras was observed, this further shows the specificity of
this assay.
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| Fig 1.
GST-PAKcrib is a p21rac activation-specific probe. (A)
Recombinant p21rac (0.2 µg) was generated and loaded in vitro with
either GDP or the nonhydrolyzable GTP analogue, GppNHp, as described in
Materials and Methods. p21rac was isolated using GST-PAKcrib and
subsequently detected by Western blot analysis. (Top) GST pull-down of
GDP-p21rac (lane 1) or GTP-p21rac (lane 2). (Middle) Coomassie staining
of the same blot showing equal GSP-PAKcrib protein in both lanes.
(Bottom) A fraction (1/40) of the sample used for the assay was probed
with anti-rac antibody to show equal amounts of p21rac were present in
both lanes. (B) BW5147 cells (107) stably retrovirally
transduced with either control virus or epitope-tagged
p21rac(V12)40 were lysed and active p21rac was isolated
using GST-PAKcrib as described in Materials and Methods. p21rac was
detected by Western blot analysis. (Top) GST pull-down of p21rac from
control (lane 1) or p21rac(V12) transfected cells (lane 2). p21rac was
detected using anti-rac antibody. (Bottom) A fraction of the lysate
used for the pull-down assay was probed with anti-p21rac. (C) (Top) GST
pull-down of p21rac from BW5147 cells stably retrovirally transduced
with control virus (lane 1), p21rac(V12) (lane 2), p21rho(V14) (lane
3), or p21ras(V12) (lane 4).40 p21rac was detected using an
antibody directed against the epitope-tag. (Middle) A fraction of the
lysate used for the pull-down was probed with antibody directed against
the epitope-tag. (Bottom) The same blot was subsequently probed with
anti-rac to demonstrate the specificity of the anti-rac antibody.
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p21rac is activated by serpentine, but not cytokine receptors in
human neutrophils.
We investigated whether GST-PAKcrib could be used to monitor p21rac-GTP
levels in human neutrophils in vivo after stimulation by the chemotaxin
fMLP. Resting neutrophils isolated from human peripheral blood were
left untreated or stimulated with fMLP for 10 seconds as indicated
(Fig 2A, top panel). p21rac was isolated using either GST or GST-PAKcrib, followed by anti-Rac immunoblotting. The rac-antibody used reacts with both rac1 and rac2, thus allowing the
simultaneous measurement of both isoforms. p21rac did not associate
with the GST alone, further demonstrating the specificity of this
assay. However, p21rac was pulled down when GST-PAKcrib was used.
Although a basal level of p21rac-GTP was observed in unstimulated
neutrophils, the amount of p21rac associating with GST-PAKcrib was
greatly enhanced when cells were stimulated with 1 µmol/L fMLP. On
the same blot, Coomassie staining was performed to show the use of
equal amounts of GST or GST-PAKcrib in all samples (Fig 2A, middle
panel). All samples contained equal amounts of p21rac (Fig 2A, bottom
panel). To investigate the kinetics of fMLP-induced p21rac activation
in human neutrophils, resting neutrophils isolated from human
peripheral blood were stimulated with fMLP for various periods of time.
p21rac was isolated with GST-PAKcrib, followed by anti-Rac
immunoblotting. Stimulation with 1 µmol/L fMLP induced an extremely
rapid and transient activation of p21rac (Fig 2B, top panel), declining
after 10 seconds. All samples contained equal amounts of p21rac (Fig
2B, middle panel) and equal amounts of GST-PAKcrib were used to
precipitate p21rac (Fig 2B, bottom panel).
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| Fig 2.
The GST-PAKcrib assay can be used to measure p21rac
activation in human neutrophils. (A) Human neutrophils were isolated as
described and stimulated with fMLP (1 µmol/L) for 10 seconds as
indicated. Cells were lysed and GST pull-down was performed using
either GST or GST-PAKcrib as described in Materials and Methods. (Top)
The isolated p21rac was detected by Western blot analysis using
anti-rac antibody. (Middle) A Coomassie staining of the blot. The
arrowheads indicate the GST and GST-PAKcrib proteins. (Bottom) A
fraction of the lysate used for the pull-down was probed with anti-Rac.
(B) Neutrophils were isolated as described and stimulated with fMLP (1 µmol/L) for the indicated time. Cells were lysed and active p21rac
was isolated using GST-PAKcrib as described in Materials and Methods.
(Top) The GST-PAKcrib-associated p21rac was detected by Western blot
analysis using anti-Rac antibody. (Middle) A fraction of the lysate
used for the pull-down was probed with anti-Rac. (Bottom) Coomassie
staining of the blot, the arrowhead indicates the GST-PAKcrib
protein.
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To investigate the effect of other stimuli on the activation of p21rac,
resting neutrophils were isolated from human peripheral blood and
stimulated with different ligands for various periods of time.
Stimulation with 1 µmol/L PAF also resulted in a rapid activation of
p21rac similar to the activation by 1 µmol/L fMLP (Fig 3A). Unexpectedly, stimulation of the
cells with GM-CSF (1010 mol/L) or TNF- (100 U/mL)
did not result in p21rac activation (Fig 3A), although these cytokines
have been shown to be potent activators of PI3K at these concentrations
in human neutrophils.6,15 To show that GM-CSF could
activate other small GTPases in these cells, we performed a
p21ras-activity assay using GST-Raf(RBD) as an activation-specific
probe.15,41 Stimulation of neutrophils with
1010 mol/L GM-CSF resulted in a transient activation
of p21ras (Fig 3B). TNF- was not able to activate p21ras (data not
shown). To determine if other human phagocytes also demonstrate
chemoattractant-mediated p21rac activation, human eosinophils were
isolated from peripheral blood as described previously.43
Stimulation of these cells for 10 seconds with fMLP or PAF also
resulted in a potent activation of p21rac (Fig 3C). Thus, it appears
that while both cytokine and chemokine receptors can activate
overlapping signaling pathways that include activation of tyrosine and
lipid kinases, only GPCRs are capable of potently activating p21rac at
physiologically relevant ligand concentrations.
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| Fig 3.
Activation of p21rac by diverse stimuli in human
neutrophils. (A) Neutrophils were isolated as described and stimulated
with either PAF (1 µmol/L), GM-CSF (1010 mol/L), or
TNF- (100 U/mL) for the indicated time. Cells were lysed and active
p21rac was isolated using GST-PAKcrib as described in Materials and
Methods. p21rac was detected by Western blot analysis. (B) Neutrophils
were stimulated with either GM-CSF (1010 mol/L) for the
indicated time or with fMLP (1 µmol/L) for 10 seconds. Cells were
lysed and active p21ras was isolated using GST-Raf1-RBD. p21ras was
detected by Western blot analysis. (C) Eosinophils were stimulated with
either fMLP (1 µmol/L) or PAF (1 µmol/L) for 10 seconds. Cells were
lysed and active p21rac was detected as above.
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fMLP activates p21rac in a dose-dependent manner independent of
GM-CSF priming.
To determine the optimal concentration at which fMLP activates p21rac,
we stimulated neutrophils with different concentrations of fMLP.
Interestingly, p21rac activation showed a bell-shaped fMLP-concentration profile, typical for serpentine receptor-induced responses such as chemotaxis, with an optimal activation between 107 and 108 mol/L fMLP.
Approximately 3% to 5% of p21rac present in whole cell lysates was
found to bind GST-PAK after 107 mol/L fMLP
stimulation of neutrophils (Fig 4A, compare
last lane with third lane). GM-CSF is known to have a strong priming
effect on both the fMLP-induced respiratory burst in human neutrophils and chemotaxis47 (and references therein). The mechanism of this priming is largely unknown. As p21rac is thought to play a role
both in the respiratory burst and cytoskeletal reorganization associated with migration, we looked at whether GM-CSF had a priming effect on the fMLP-induced activation of p21rac. Neutrophils were primed with 1010 mol/L GM-CSF before stimulation
with either 108 mol/L fMLP or a suboptimal dose of
1010 mol/L fMLP. We found no enhanced or extended
activation when cells were stimulated with 108 mol/L
fMLP (Fig 4B, top panel). Similarly, GM-CSF did not enhance the
sensitivity of the cells toward a suboptimal dose of
1010 mol/L fMLP (Fig 4B, bottom panel). Thus, the
mechanism of GM-CSF-mediated priming of the respiratory burst does not
appear to involve activation of p21rac.
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| Fig 4.
Effect of GM-CSF on fMLP-induced p21rac activation. (A)
Neutrophils were isolated as described and stimulated with the
indicated final concentrations of fMLP for 10 seconds. p21rac
activation was determined as described in the legend to Fig 1. One
percent of a total cell lysate was used for comparison with the amount
of activated p21rac. (B) Neutrophils were pretreated for 20 minutes
with 1010 mol/L GM-CSF after stimulation with either
108 mol/L fMLP (top) or 1010 mol/L fMLP
(bottom) for the time indicated.
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fMLP-induced p21rac activation is not inhibited by kinase inhibitors.
We used several protein and lipid kinase inhibitors to determine the
mechanism by which p21rac is activated by fMLP in human neutrophils.
First, resting human neutrophils were treated with the broad
specificity tyrosine kinase inhibitors, genistein and erbstatin.
Engagement of fMLP receptors has been shown to induce tyrosine
phosphorylation-mediated signaling events in neutrophils.48 These inhibitors effectively block or greatly reduce tyrosine phosphorylation in human neutrophils in response to diverse
stimuli.16 Pretreatment of the cells with 100 µmol/L
genistein or erbstatin had no effect on fMLP-induced p21rac activation
(Fig 5A, left panel). Studies in various
cell lines have suggested that p21rac activation may lie downstream of
the lipid kinase PI3K.28,49,50 Furthermore, inhibition of
PI3K with the specific inhibitor, LY294002, has been shown to block the
fMLP-induced respiratory burst in neutrophils completely51
(see also Fig 5B). As fMLP induces PI3K activation in human
neutrophils,52 we investigated whether this kinase may play
a role in the activation of p21rac. Addition of LY294002 to human
neutrophils at concentrations that inhibit PI3K53 had no
effect on p21rac activation (Fig 5A, right panel). The same was
observed for wortmannin, another potent PI3K inhibitor (data not
shown). However, as expected, LY294002 completely blocked the
fMLP-induced respiratory burst (Fig 5B).
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| Fig 5.
Effect of tyrosine kinase inhibitors on p21rac
activation. (A) Neutrophils were pretreated for 20 minutes with 100 µmol/L genistein, 100 µmol/L erbstatin, 50 µmol/L PD098059, 10 µmol/L SB203580, 50 µmol/L PP1, or 10 µmol/L LY294002 after
stimulation with fMLP (1 µmol/L) for 10 seconds. (B) Neutrophils were
either untreated (a), pretreated with 10 µmol/L LY294002 for 20 minutes and then GM-CSF (1010 mol/L) for 20 minutes (b),
or with GM-CSF (1010 mol/L) for 20 minutes (c). At the
time indicated by the arrowhead, samples were stimulated with fMLP (1 µmol/L). Oxygen consumption was measured using a Clark oxygen
electrode as previously described.
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Using a variety of specific pharmacologic inhibitors including the ERK
inhibitor, PD098059, the p38 mitogen activated protein kinase (MAPK)
inhibitor, SB203580, and the Src inhibitor, PP1, we investigated the
role of these kinases in mediating p21rac activation. We and others
have previously shown these inhibitors to be potent and specific in
human neutrophils at these concentrations.15,54 None of
these compounds had an inhibitory effect on the fMLP-induced p21rac
activation (Fig 5A, right panel). These data suggest a novel mechanism
for p21rac activation independent of tyrosine phosphorylation or PI3K.
Furthermore, it indicates that the mechanism by which inhibition of
PI3K results in abrogation of superoxide production does not involve
p21rac, but apparently another component of the NADPH oxidase system.
p21rac activation is independent of changes in intracellular
Ca2+.
Although the fMLP receptor has been shown to activate a variety of
tyrosine-phosphorylation-mediated signaling events, activation of
protein kinase C (PKC) and an increase in intracellular
free Ca2+ ([Ca2+]i) have also
been described.55 Stimulation of neutrophils with fMLP
results in a very rapid and transient increase in intracellular free
[Ca2+]i that correlates with the activation
of p21rac (Fig 6A). To investigate a
possible role for Ca2+ and/or PKC in fMLP-induced p21rac
activation, we depleted neutrophils of Ca2+ by pretreatment
with 1 mmol/L EGTA, 1.5 µmol/L indo-1/AM, and 100 nmol/L
thapsigargin. This causes a decrease of
[Ca2+]i to less than 10 nmol/L, and fMLP
stimulation no longer induces an increase in
[Ca2+]i (Fig 6B).
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| Fig 6.
Effect of Ca2+ depletion on p21rac
activation. (A) Neutrophils were stimulated with fMLP
(108 mol/L) for the time indicated. p21rac activation
was determined as previously described. In cells from the same donor
change in [Ca2+]i was measured after
stimulation. The arrowhead indicates addition of fMLP
(108 mol/L). (B) Neutrophils were
Ca2+-depleted before stimulation with fMLP
(108 mol/L). p21rac activation and
[Ca2+]i were measured as described above.
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As shown in Fig 6B, Ca2+-depletion did not affect the
fMLP-induced p21rac activation. It has been shown for the activation of the Rap1 small GTPase in neutrophils that although Ca2+ is
not essential for the activation of Rap1 by fMLP, Ca2+
influx induced with ionomycin or thapsigargin resulted in Rap1 activation.17 Therefore, we investigated whether ionomycin
or thapsigargin could activate p21rac. As shown in
Fig 7A, neither ionomycin nor thapsigargin
induced p21rac activation compared with an fMLP (108
mol/L) control. Surprisingly, PMA, an activator of PKC that strongly induces the respiratory burst in human neutrophils, did not induce p21rac activation. As a control, we measured the respiratory burst induced by PMA in cells from the same donor. As shown in Fig 7B, PMA
strongly activated the respiratory burst under conditions that did not
activate p21rac. This demonstrates that superoxide production per se in
human neutrophils can occur independently of p21rac activation.
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| Fig 7.
PMA activates the respiratory burst independently of
p21rac. (A) Neutrophils were stimulated with fMLP (108
mol/L), PMA (100 ng/mL), ionomycin (500 nmol/L), or thapsigargin (100 nmol/L) for the time indicated. p21rac activation was determined as
described. (B) In cells from the same donor, oxygen consumption was
measured after stimulation with PMA (100 ng/mL).
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Role of heterotrimeric G-protein -subunits in
fMLP-mediated activation of p21rac.
fMLP and PAF both act via serpentine receptors coupled to
heterotrimeric G-protein subunits. Recently, a conserved motif was found between a GEF for Rho GTPases (p115RhoGEF) and members of the
Regulators of G protein Signaling (RGS) family.56,57 It is
suggested that p115RhoGEF links the inactivation of G12
and G13 proteins to the activation of Rho through the
direct association with the activated G13 subunits. To
determine whether direct activation of heterotrimeric G protein
-subunits can in itself play a role in the activation of p21rac, we
stimulated cells with AlF4, which causes
specific activation of the -subunits of heterotrimeric G-proteins.58 Indeed, AlF4
has previously been demonstrated to induce actin-polymerization via
G-proteins in human neutrophils.59 We found that
AlF4 activates p21rac with similar
kinetics, as does fMLP (Fig 8). This
suggests, although indirectly, that there is a potential direct role
for G-subunits in the activation of p21rac by serpentine receptors.
View larger version (27K):
[in this window]
[in a new window]
| Fig 8.
p21rac can be activated AlF4.
Neutrophils were stimulated with AlF4 as
described in Materials and Methods for the indicated times. As a
control, neutrophils were stimulated with fMLP (108
mol/L) for 10 seconds. p21rac activation was determined as previously
described.
|
|
| |
DISCUSSION |
This is the first report to describe the activation of p21rac by
chemoattractants in vivo. Activation of p21rac results in the exchange
of bound GDP for GTP. Conventionally, activation of small G-proteins is
measured by the immunoprecipitation of the protein and measurement of
GTP loading. However, as endogenous p21rac cannot be immunoprecipitated
from cell lines or primary cells,28 direct measurement of
the GTP loading of p21rac has not been possible. We therefore used a
novel assay to measure p21rac activation in vivo. This assay is based
on the finding that GTP-p21rac specifically associates with the
Cdc42/p21rac interactive binding (CRIB) motif of PAK, whereas
GDP-p21rac does not (Fig 1).34,35 In stably retrovirally
transduced BW5137 cells, p21rac(V12), but not p21ras(V12) or
p21rho(V14), associated with GST-PAKcrib, demonstrating the specificity
of this interaction (Fig 1). Although the expression level of
p21rac(V12) was extremely low as compared with the endogenous p21rac,
p21rac(V12) was shown to potently bind GST-PAKcrib. We believe this is
not a consequence of aspecific binding of GDP-p21rac to the
GST-PAKcrib, as we observed no interaction between the recombinant
GDP-p21rac and GST-PAKcrib, nor did a GST control pull-down any p21rac
from a neutrophil lysate (see Figs 1A and 2A). The low background
binding of endogenous p21rac that was nevertheless observed is probably
due to serum activation of the endogenous p21rac.
An extremely rapid activation of p21rac was observed on stimulation of
human neutrophils with the chemoattractants fMLP or PAF, with an
optimal activation within 5 to 10 seconds. Previous studies in cell
lines have suggested that activation of p21rac lies downstream of
PI3K.28,49,50 Unexpectedly, stimulation of neutrophils with
TNF- or GM-CSF had no effect on p21rac activity, although we and
others have previously reported that both of these cytokines are potent
activators of the lipid kinase PI3K in neutrophils.6,15 This suggests that activation of PI3K per se is not sufficient to
induce p21rac activation in neutrophils. This is supported by a recent
report, showing that the dissociation of gelsolin from actin filaments,
which initiates actin polymerization, is regulated by p21rac
independently of PI3K.60 Recent reports have also indicated
that PI3K can indeed act downstream of p21rac. Inhibition of PI3K in
cells expressing dominant active p21rac blocked the rac-induced
spreading and the formation of actin structures,61 and
bombesin-stimulated membrane ruffling, which requires p21rac, is not
blocked by wortmannin.62 Furthermore, a direct association between GTP-p21rac and PI3K has been reported in neutrophils, which
caused an increase in PI3K activity.63 In addition,
tyrosine kinase inhibitors, genistein and erbstatin, and specific
inhibitors of MEK, p38 MAPK, Src, and PI3K had no effect on the
fMLP-induced p21rac activation, suggesting that in human neutrophils
fMLP-induced p21rac activation is via an alternative pathway.
During the preparation of this report, an article reported that in
transfected COS-7 cells expressing the fMLP receptor, fMLP-induced cortical actin reorganization was blocked by dominant-negative mutants
of PI3K, Vav, and p21rac.50 These data suggests that fMLP activates p21rac via PI3K-mediated activation of Vav. This is
in contrast to the data reported herein concerning the mechanisms of
fMLP-mediated p21rac activation in neutrophils. One explanation could
be that expression of the fMLP receptor in COS cells, a cell that does
not normally respond to fMLP, results in the utilization of signaling
pathways not normally used for p21rac activation. Indeed, activation of
fMLP receptors overexpressed in RBL cells has been shown to cause an
abnormally sustained activation profile relative to cells endogenously
expressing the receptor.64
p21rac is known to regulate actin assembly in a variety of cells,
resulting in cell spreading and the formation of lamellipodia that may
play a role in cell migration (see Hall65). It is
interesting to note that, while GM-CSF and TNF- promote chemokinesis
(random cell migration) and do not activate p21rac, PAF and fMLP are
strong chemoattractants (directed movement of cells) and do activate p21rac15 (Figs 2 and 3). Possibly the activation of p21rac
plays an important role in the regulation of chemotaxis rather than random migration, suggesting interesting mechanistic variations between
these two methods of cell movement. Indeed, it has been demonstrated
that migration of fibroblasts towards platelet-derived growth factor
(PDGF) is inhibited by expression of dominant-negative N17rac1.66 Furthermore, in macrophage cell lines,
microinjection of dominant-negative N17rac1 inhibited CSF-1-induced
polarization and migration.67
Another cellular response that has been linked to the activation of
p21rac is the production of superoxide.21,22 Studies in
cell-free extracts have suggested that p21rac is necessary for the
assembly of the NADPH oxidase complex.23-26 The involvement of p21rac in the oxidase is also supported by the parallel reduction of
p21rac expression and NADPH oxidase activation in Epstein-Barr virus
(EBV)-transformed B cells cultured in the presence of
antisense p21rac oligonucleotides.27 Indeed, interaction of
p21rac with the p67phox component of the NADPH oxidase system has also
been shown.68,69 This association is then thought to target
the p67phox and associated p47phox proteins to the membrane where another component of the oxidase system, flavocytochrome b558, is
present. In a patient suffering from chronic granulomatous disease, a
single amino acid deletion was found in p67phox, which disrupts the
interaction with p21rac.70 Neutrophils isolated from this
patient failed to produce superoxide. Furthermore, overexpression of
the dominant negative form of p21rac in HL60 cells inhibited the
production of superoxide by these cells.24 We show that although inhibition of PI3K does not inhibit p21rac, it completely blocks the respiratory burst in neutrophils. This again suggests that
p21rac is not downstream of PI3K in fMLP-stimulated neutrophils, but
may act in parallel with this kinase to activate production of
superoxide. fMLP stimulation of neutrophils results in changes in
intracellular-free [Ca2+] levels,55 which are
necessary for the respiratory burst to occur.71 Although
the time frame of the fMLP-induced p21rac activation and the increase
in intracellular Ca2+ are similar, Ca2+
depletion did not affect the fMLP-induced p21rac activation, showing
that p21rac can be activated independently of changes in
[Ca2+]i. Similarly, Ca2+-influx
induced by ionomycin or calcium release from intracellular stores
induced by thapsigargin did not induce p21rac activation. Furthermore,
PMA failed to activate p21rac, although in the same cells, PMA leads to
a strong activation of the respiratory burst. This demonstrates that it
is possible to activate superoxide production independently of p21rac
using certain stimuli. In support of this finding, Philips et
al72 showed that although fMLP stimulation of human
neutrophils induced a transient increase in membrane-bound p21rac,
stimulation with PMA did not cause p21rac translocation. Furthermore,
in a cell-free assay, bacterially expressed unprenylated p21rac, which
is not membrane-associated, can effectively activate the oxidase
system25 calling into question both the necessity for
p21rac to be translocated to the membrane fraction and the need for
GTPase function. Thus, the precise mechanism of action of p21rac in the
activation of the respiratory burst still needs to be determined. It
has been reported that GTP loading of p21rac is necessary for the
interaction between p21rac and p67phox.68 On the other
hand, Bromberg et al23 have shown that GDP-p21rac complexed
to the GDP dissociation inhibitor for p21Rho (Rho GDI) can activate the
NADPH oxidase system in vitro. It is tempting to speculate that in
neutrophils, p21rac does not need to be activated (GTP-bound) for the
assembly and activation of the NADPH oxidase complex, but rather in a
GDP-bound state complexed to Rho GDI.
Because fMLP-induced p21rac activation is not mediated by tyrosine
kinases or induced by changes in [Ca2+]i, the
mechanism of its activation remains elusive. Recently, a novel
mechanism was reported for the activation of small GTPases by
serpentine receptors. A conserved motif found between a GEF for Rho
GTPases (p115RhoGEF) and members of the RGS family suggested that
p115RhoGEF links the inactivation of G12 and
G13 proteins to the activation of Rho through the direct
association with the activated G-subunits.56,57
Furthermore, a direct association was shown between G13
and p115RhoGEF. Indeed, we found a similar homology between the
exchange factor for p21rac, Vav, and several members of the RGS family
(not shown). To explore the possibility that G-subunits may directly
activate p21rac, human neutrophils were treated with
AlF4. It is known that
AlF4 causes specific activation of the
subunits of heterotrimeric G-proteins by binding to
G-GDP resulting in a GTP-like transition state.58
Previously, it has been shown that AlF4
treatment of human neutrophils results in a pronounced increase in
F-actin content.59 Interestingly,
AlF4 treatment also resulted in the
rapid and transient activation of p21rac with a time course that
closely resembles that observed for fMLP and PAF (Fig 8). Our data
suggest that this may be due to G-protein activation of p21rac.
Although indirect, these data add weight to the idea that that
activation of Vav by serpentine receptors may occur via an alternative
direct mechanism requiring neither tyrosine phosphorylation nor
3-phosphorylated lipids, but rather occurring via a direct
G-mediated mechanism.
| |
ACKNOWLEDGMENT |
We thank Tim Reid for the generation of GST-PAKcrib and Reza Ahmadian
for the generation of recombinant p21rac and technical assistance with
the GDP/GTP loading. Finally, we thank Laura M'Rabet for helpful
discussions and Leo Houben for technical assistance.
| |
FOOTNOTES |
Submitted October 12, 1998; accepted April 12, 1999.
Supported by GlaxoWellcome b.v., The Netherlands.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Paul J. Coffer, PhD,
Department of Pulmonary Diseases, University Hospital Utrecht, G03.550,
Heidelberglaan 100, 3584 CX Utrecht, The Netherlands; e-mail:
P.Coffer{at}hli.azu.nl.
| |
REFERENCES |
1.
Haslett C, Savill JS, Meagher L:
The neutrophil.
Curr Opin Immunol
2:10, 1989[Medline]
[Order article via Infotrieve]
2.
Bokoch GM:
Regulation of phagocyte function by low-molecular weight GTP-binding proteins.
Eur J Haematol
51:313, 1993[Medline]
[Order article via Infotrieve]
3.
DeLeo FR, Quinn MT:
Assembly of the phagocyte NADPH oxidase: Molecular interaction of oxidase proteins.
J Leukoc Biol
60:677, 1996[Abstract]
4.
Sandborg RR, Smolen JE:
Early biochemical events in leukocyte activation.
Lab Invest
59:300, 1988[Medline]
[Order article via Infotrieve]
5.
Sha'afi RI, Molski TF:
Activation of the neutrophil.
Prog Allergy
42:1, 1988
6.
Al-Shami A, Bourgoin SG, Naccache PH:
Granulocyte-macrophage colony-stimulating factor activated signaling pathways in human neutrophils.
Blood
89:1035, 1997[Abstract/Free Full Text]
7.
Arcaro A, Wymann MP:
Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: The role of phosphatidyl 3,4,5-triphosphate in neutrophil responses.
Biochem J
296:297, 1993
8.
Fialkow L, Chan CK, Rotin D, Grinstein S, Downey GP:
J Biol Chem
269:31234, 1994[Abstract/Free Full Text]
9.
Gomez-Cambronero J, Huang C-K, Gomez-Cambronero TM, Waterman WH, Becker EL, Sha'afi RI:
Granulocyte-macrophage colony-stimulating factor-induced protein tyrosine phosphorylation of microtubule-associated protein kinase in human neutrophils.
Proc Natl Acad Sci USA
89:7551, 1992[Abstract/Free Full Text]
10.
Nahas N, Molski TF, Fernandez GA, Sha'afi RI:
Tyrosine phosphorylation and activation of a new mitogen-activated protein (MAP)-kinase cascade in human neutrophils stimulated with various agonists.
Biochem J
318:247, 1996
11.
Okada T, Sakuma L, Fukui Y, Hazeki O, Ui M:
Blockage of chemotactic peptide induced stimulation of neutrophils by wortmannin as a result of selective inhibition of phosphatidylinositol 3-kinase.
J Biol Chem
269:3563, 1994[Abstract/Free Full Text]
12.
Pillinger MH, Feokistov AS, Capodici C, Solitar B, Levy J, Oei TT, Philips MR:
Mitogen-activated protein kinase and neutrophils an enucleate neutrophil cytoplasts: Evidence for regulation of cell-cell adhesion.
J Biol Chem
271:12049, 1996[Abstract/Free Full Text]
13.
Torres M, Hall FL, O'Neill K:
Stimulation of human neutrophils with formyl-methionyl-leucyl-phenylalanine induces tyrosine phosphorylation of two distinct mitogen-activated protein kinases.
J Immunol
150:1563, 1993[Abstract]
14.
Waterman WH, Sha'afi RI:
Effects of granulocyte-macrophage colony-stimulating factor and tumour necrosis factor-alpha on tyrosine phosphorylation and activation of mitogen-activated protein kinases in human neutrophils.
Biochem J
307:39, 1995
15.
Coffer PJ, Geijsen N, M'Rabet L, Schweizer RC, Maikoe T, Raaijmakers JAM, Lammers J-WJ, Koenderman L:
Comparison of the roles of mitogen-activated protein kinase kinase and phosphatidylinositol 3-kinase signal transduction in neutrophil effector function.
Biochem J
329:121, 1998
16.
Zheng L, Eckerdal J, Dimitrijevic I, Andersson T:
Chemotactic peptide-induced activation of Ras in human neutrophils is associated with inhibition of p120-GAP Activity.
J Biol Chem
272:23448, 1997[Abstract/Free Full Text]
17.
M'Rabet L, Coffer P, Zwartkruis F, Franke B, Segal AW, Koenderman L, Bos JL:
Activation of the small GTPase Rap1 in human neutrophils.
Blood
92:2133, 1998[Abstract/Free Full Text]
18.
Bokoch GM:
Regulation of the human neutrophil NADPH oxidase by the rac GTP-binding proteins.
Curr Opin Cell Biol
6:212, 1994[Medline]
[Order article via Infotrieve]
19.
Bokoch GM:
Chemoattractant signaling and leukocyte activation.
Blood
86:1649, 1995[Free Full Text]
20.
Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A:
The small GTP-binding protein regulates growth-factor induced membrane ruffling.
Cell
70:401, 1992[Medline]
[Order article via Infotrieve]
21.
Abo A, Pick E, Hall A, Totty N, Teahan CG, Segal AW:
Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1.
Nature
353:668, 1991[Medline]
[Order article via Infotrieve]
22.
Knaus UG, Hayworth PG, Kinsella BT, Curnutte JT, Bokoch GM:
Purification and characterisation of rac2. A cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase.
J Biol Chem
267:23575, 1992[Abstract/Free Full Text]
23.
Bromberg Y, Shani E, Joseph G, Gorzalczany Y, Sperling O, Pick E:
The GDP-bound form of the small G protein p21rac1 is a potent activator of the superoxide-forming NADPH oxidase of macrophages.
J Biol Chem
269:7055, 1994[Abstract/Free Full Text]
24.
Gabig TG, Crean CD, Mantel PL, Rosli R:
Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL-60 cell NADPH oxidase activation.
Blood
85:804, 1995[Abstract/Free Full Text]
25.
Heyworth PG, Knaus UG, Settleman J, Curnutte JT, Bokoch GM:
Regulation of NADPH oxidase by Rac GTPase activating protein(s).
Mol Biol Cell
4:1217, 1993[Abstract]
26.
Kreck ML, Uhlenger DJ, Tyagi SR, Inge KL, Lambeth JD:
Participation of the small molecular weight GTP-binding protein Rac1 in cell-free activation and assembly of the respiratory burst oxidase.
J Biol Chem
269:4161, 1994[Abstract/Free Full Text]
27.
Dorseuil O, Vasquez A, Lang P, Bertoglio J, Garcon G, Leca G:
Inhibition of superoxide production in B-lymphocytes by Rac antisense oligonucleotides.
J Biol Chem
267:20540, 1992[Abstract/Free Full Text]
28.
Hawkins PT, Eguinoa A, Qiu RG, Stokoe D, Cooke FT, Walters R, Wennstrom S, Claesson-Welsh L, Evans T, Symons M:
PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase.
Curr Biol
5:393, 1995[Medline]
[Order article via Infotrieve]
29.
Crespo P, Schuebel KE, Ostrom AA, Gutkind JS, Bustelo XR:
Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product.
Nature
385:169, 1997[Medline]
[Order article via Infotrieve]
30.
Habets GG, Scholtes EH, Zuydgeest D, van der Kammen RA, Stam JC, Berns A, Collard JG:
Identification of an invasion-inducing gene, Tiam-1, that encodes a protein with homology for GDP-GTP exchangers for Rho-like proteins.
Cell
77:537, 1994[Medline]
[Order article via Infotrieve]
31.
Han J, Luby-Phelps K, Das B, Shu X, Xia Y, Mosteller RD, Krishna UM, Falck JR, White MA, Broek D:
Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav.
Science
279:558, 1998[Abstract/Free Full Text]
32.
Matsuguchi T, Inhorn RC, Carlesso N, Xu G, Druker B, Griffin JD:
Tyrosine phosphorylation of p95Vav in myeloid cells is regulated by GM-CSF, IL-3 and steel factor and is constitutively increased by p210Bcr/Abl.
EMBO J
14:257, 1995[Medline]
[Order article via Infotrieve]
33.
Sander EE, van Delft S, ten Klooster JP, Reid T, van der Kammen RA, Michiels F, Collard JG:
Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase.
J Cell Biol
143:1385, 1998[Abstract/Free Full Text]
34.
Manser E, Leung T, Salihuddin H, Zhao Z-S, Lim L:
A brain serine/threonine protein kinase activated by Cdc42 and Rac1.
Nature
367:40, 1994[Medline]
[Order article via Infotrieve]
35.
Manser E, Loo T-H, Koh C-G, Zhao Z-S, Chen X-Q, Tan L, Tan I, Leung T, Lim L:
PAK kinases are directly coupled to the PIX family of nucleotide exchange factors.
Mol Cell
1:183, 1998[Medline]
[Order article via Infotrieve]
36.
Thompson G, Owen D, Chalk PA, Lowe PN:
Delineation of the Cdc42/Rac-binding domain of p21-activated kinase.
Biochemistry
37:7885, 1998[Medline]
[Order article via Infotrieve]
37.
Koenderman L, Kok PT, Hamelink ML, Verhoeven AJ, Bruijnzeel PL:
An improved method for the isolation of eosinophilic granulocytes from peripheral blood of normal individuals.
J Leukoc Biol
44:79, 1988[Abstract]
38.
Dewald B, Baggiolini M:
Activation of NADPH oxidase in human neutrophils. Synergism between fMLP and neutrophil products PAF and LTB4.
Biochem Biophys Res Commun
128:297, 1985[Medline]
[Order article via Infotrieve]
39.
Weisbart RH, Kwan L, Golde DW, Gasson JC:
Human GM-CSF primes neutrophil for enhanced oxidative metabolism in response to the major physiological chemoattractants.
Blood
69:18, 1987[Abstract/Free Full Text]
40.
Stam JC, Michiels F, van der Kammen RA, Moolenaar WH, Collard JG:
Invasion of T-lymphoma cells: Cooperation between Rho family GTPases and lysophospholipid receptor signaling.
EMBO J
17:4066, 1998[Medline]
[Order article via Infotrieve]
41.
de Rooij J, Bos JL:
Minimal ras-binding domain of Raf1 can be used as an activation-specific probe for Ras.
Oncogene
14:623, 1997[Medline]
[Order article via Infotrieve]
42.
Vetter IR, Nowak C, Nishimoto T, Kuhlmann J, Wittinghofer A:
Structure of a Ran-binding domain complexed with Ran bound to a GTP analogue: Implications for nuclear transport.
Nature
398:39, 1999[Medline]
[Order article via Infotrieve]
43.
Koenderman L, Yazdanbakhsh M, Roos D, Verhoeven AJ:
Dual mechanisms in priming of the chemoattractant-induced respiratory burst in human granulocytes. A Ca2+-dependent and a Ca2+-independent route.
J Immunol
142:623, 1989[Abstract]
44.
Weening RS, Roos D, Loos JA:
Oxygen consumption of phagocytizing cells in human leukocyte an granulocyte preparations: A comparative study.
J Lab Clin Med
83:570, 1974[Medline]
[Order article via Infotrieve]
45.
Knaus UG, Morris S, Dong H-J, Chernoff J, Bokoch GM:
Regulation of human leukocyte p21-activated kinases through G protein-coupled receptors.
Science
269:221, 1995[Abstract/Free Full Text]
46.
Ding J, Knaus UG, Lian JP, Bokoch GM, Badwey JA:
The renaturable 69- and 63-kDa protein kinases that undergo rapid activation in chemoattractant-stimulated guinea pig neutrophils are p21-activated kinases.
J Biol Chem
271:24869, 1996[Abstract/Free Full Text]
47.
Coffer PJ, Koenderman L:
Granulocyte signal transduction and priming: cause without effect?
Immunol Lett
57:27, 1997[Medline]
[Order article via Infotrieve]
48.
Ptasznik A, Traynor-Kaplan A, Bokoch GM:
G protein-coupled chemoattractant receptors regulate Lyn tyrosine kinase. Shc adapter protein complexes.
J Biol Chem
270:19969, 1995[Abstract/Free Full Text]
49.
Kotani K, Hara K, Kotani K, Yonezawa K, Kasuga M:
Phosphoinositide 3-kinase as an upstream regulator of the small GTP-binding protein Rac in the insulin signaling of membrane ruffling.
Biochem Biophys Res Commun
208:985, 1995[Medline]
[Order article via Infotrieve]
50.
Ma AD, Metjian A, Bagrodia S, Taylor S, Abrahams CS:
Cytoskeletal reorganisation by G protein-coupled receptors is dependent on phosphoinositide 3-kinase , a Rac guanosine exchange factor, and Rac.
Mol Cell Biol
18:4744, 1998[Abstract/Free Full Text]
51.
Vlahos CJ, Matter WF, Brown RF, Traynor-Kaplan AE, Heyworth PG, Prossnitz ER, Ye RD, Marder P, Schelm JA, Rothfuss KJ:
Investigation of neutrophil signal transduction using a specific inhibitor of phosphatidylinositol 3-kinase.
J Immunol
154:2413, 1995[Abstract]
52.
Stephens LR, Hughes KT, Irvine RF:
Pathway of phosphatidylinositol(3,4,5)-trisphopshate synthesis in activated neutrophils.
Nature
351:33, 1991[Medline]
[Order article via Infotrieve]
53.
Vlahos CJ, Matter WF, Hui KY, Brown RF:
A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002).
J Biol Chem
269:5241, 1994[Abstract/Free Full Text]
54.
Zu YI, Gilchrist A, Fernandez GA, Vazquez-Abad D, Kreutzer DL, Huiang CK, Sha'afi RI:
p38 mitogen-activated protein kinase activation is required for human neutrophil function triggered by TNF or fMLP stimulation.
J Immunol
160:1982, 1998[Abstract/Free Full Text]
55.
O'Flaherty JT, Jacobson DP, Redman JF, Rossi AG:
Translocation of protein kinase C in polymorphonuclear neutrophils. Regulation by cytosolic Ca2+-independent and Ca2+-dependent mechanisms.
J Biol Chem
265:9146, 1990[Abstract/Free Full Text]
56.
Hart MJ, Jiang X, Kozasa T, Roscoe W, Singer WD, Gilman AG, Sternweis PC, Bollag G:
Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by G13.
Science
280:2112, 1998[Abstract/Free Full Text]
57.
Kozasa T, Jiang X, Hart MJ, Sternweis PM, Singer WD, Gilman AG, Bollag G, Sternweis PC:
p115 RhoGEF a GTPase activating protein for G12 and G13.
Science
280:2109, 1998[Abstract/Free Full Text]
58.
Kahn RA:
Fluoride is not an activator of the smaller (20-25 kDa) GTP-binding proteins.
J Biol Chem
266:15595, 1991[Abstract/Free Full Text]
59.
Bengtsson T, Sarndahl E, Stendahl O, Andersson T:
Involvement of GTP-binding proteins in actin-polymerization in human neutrophils.
Proc Natl Acad Sci USA
87:2921, 1990[Abstract/Free Full Text]
60.
Arcaro A:
The small GTP-binding protein Rac promotes the dissociation of gelsolin from actin filaments in neutrophils.
J Biol Chem
273:805, 1998[Abstract/Free Full Text]
61.
Keely PJ, Westwick JK, Whitehead IP, Der CJ, Parise LV:
Cdc42 and Rac1 induce integrin-mediated cell-motility and invasiveness through PI(3)K.
Nature
390:632, 1997[Medline]
[Order article via Infotrieve]
62.
Nobes CD, Hawkins P, Stephens L, Hall A:
Activation of the small GTP-binding proteins Rho and Rac by growth factor receptors.
J Cell Sci
108:225, 1995[Abstract]
63.
Bokoch GM, Vlahos CJ, Wang Y, Knaus UG, Traynor-Kaplan AE:
Rac GTPase interacts specifically with phosphatidylinositol 3-kinase.
Biochem J
315:775, 1996
64.
Hall AL, Wilson BS, Pfeiffer JR, Oliver JM, Sklar LA:
Relationship of ligand-receptor dynamics to actin polymerisation in RBL-2H3 cells transfected with the human formyl peptide receptor.
J Leukoc Biol
62:535, 1997[Abstract]
65.
Hall A:
Rho GTPases and the actin cytoskeleton.
Science
279:509, 1998[Abstract/Free Full Text]
66.
Anand-Apte B, Zetter BR, Viswanathan A, Qui RG, Chen J, Ruggieri R, Symons M:
Platelet-derived growth factor and fibronectin-stimulated migration are differentially regulated by the Rac and extracellular-regulated kinase pathways.
J Biol Chem
272:30688, 1997[Abstract/Free Full Text]
67.
Allen WE, Zicha D, Ridley AJ, Jones GE:
A role for Cdc42 in macrophage chemotaxis.
J Cell Biol
141:1147, 1998[Abstract/Free Full Text]
68.
Diekmann D, Abo A, Segal AW, Hall A:
Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity.
Science
265:531, 1994[Abstract/Free Full Text]
69.
Dorseuil O, Reibel L, Bokoch GM:
The Rac target NADPH oxidase p67phox interacts preferentially with Rac2 rather than Rac1.
J Biol Chem
271:83, 1996[Abstract/Free Full Text]
70.
Leusen JH, de Klein A, Hilarius PM, Ahlin A, Palmblad J, Smith CI, Diekmann D, Hall A, Verhoeven AJ, Roos D:
Disturbed interaction with p21-rac with p67-phox causes chronic granulomatous disease.
J Exp Med
184:1243, 1996[Abstract/Free Full Text]
71.
Foyouzi-Youssefi R, Petersson F, Lew DP, Krause KH, Nusse O:
Chemoattractant-induced respiratory burst: Increases in cytosolic Ca2+ concentrations are essential and synergise with a kinetically distinct signal.
Biochem J
322:709, 1997
72.
Philips MR, Feoktistov A, Pillinger MH, Abramson SB:
Translocation of p21rac from cytosol to plasma membrane is neither necessary nor sufficient for neutrophil NADPH oxidase activity.
J Biol Chem
270:11514, 1995[Abstract/Free Full Text]
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|
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|
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[Full Text]
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[Full Text]
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[Abstract]
[Full Text]
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|
|
|
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[Full Text]
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|
|
|
|
|
|
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|
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|
|
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|
|
|
|
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|
|
|
|
|
|
|
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|
|
|
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|
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[Full Text]
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|
|
|
|
|
|
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[Full Text]
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|
|
|
|
|
|
|
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[Full Text]
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|
|
|
|
|
|
|
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October 16, 2002;
100(9):
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[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Betson, E. Lozano, J. Zhang, and V. M. M. Braga
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J. Biol. Chem.,
September 27, 2002;
277(40):
36962 - 36969.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M Pelletier and D Girard
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August 1, 2002;
21(8):
415 - 420.
[Abstract]
[PDF]
|
|
|
|
|
|
|
H. Kanazawa, K. Ohsawa, Y. Sasaki, S. Kohsaka, and Y. Imai
Macrophage/Microglia-specific Protein Iba1 Enhances Membrane Ruffling and Rac Activation via Phospholipase C-gamma -dependent Pathway
J. Biol. Chem.,
May 24, 2002;
277(22):
20026 - 20032.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. van den Eijnde, M. J. B. van den Hoff, C. P. M. Reutelingsperger, W. L. van Heerde, M. E. R. Henfling, C. Vermeij-Keers, B. Schutte, M. Borgers, and F. C. S. Ramaekers
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J. Cell Sci.,
March 12, 2002;
114(20):
3631 - 3642.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Nijhuis, J.-W. J Lammers, L. Koenderman, and P. J. Coffer
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J. Leukoc. Biol.,
January 1, 2002;
71(1):
115 - 124.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M.-P. Gratacap, B. Payrastre, B. Nieswandt, and S. Offermanns
Differential Regulation of Rho and Rac through Heterotrimeric G-proteins and Cyclic Nucleotides
J. Biol. Chem.,
December 14, 2001;
276(51):
47906 - 47913.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Adachi, R. Vita, S. Sannohe, S. Stafford, R. Alam, H. Kayaba, and J. Chihara
The Functional Role of Rho and Rho-Associated Coiled-Coil Forming Protein Kinase in Eotaxin Signaling of Eosinophils
J. Immunol.,
October 15, 2001;
167(8):
4609 - 4615.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. M. Chinnaiyan, M. Huber-Lang, C. Kumar-Sinha, T. R. Barrette, S. Shankar-Sinha, V. J. Sarma, V. A. Padgaonkar, and P. A. Ward
Molecular Signatures of Sepsis : Multiorgan Gene Expression Profiles of Systemic Inflammation
Am. J. Pathol.,
October 1, 2001;
159(4):
1199 - 1209.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. Alblas, L. Ulfman, P. Hordijk, and L. Koenderman
Activation of RhoA and ROCK Are Essential for Detachment of Migrating Leukocytes
Mol. Biol. Cell,
July 1, 2001;
12(7):
2137 - 2145.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. P. van Nieuw Amerongen and V. W.M. van Hinsbergh
Cytoskeletal Effects of Rho-Like Small Guanine Nucleotide-Binding Proteins in the Vascular System
Arterioscler Thromb Vasc Biol,
March 1, 2001;
21(3):
300 - 311.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. P. Lian, L. Crossley, Q. Zhan, R. Huang, P. Coffer, A. Toker, D. Robinson, and J. A. Badwey
Antagonists of Calcium Fluxes and Calmodulin Block Activation of the p21-Activated Protein Kinases in Neutrophils
J. Immunol.,
February 15, 2001;
166(4):
2643 - 2650.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. A. Jefferies and L. A. J. O'Neill
Rac1 Regulates Interleukin 1-induced Nuclear Factor kappa B Activation in an Inhibitory Protein kappa Balpha -independent Manner by Enhancing the Ability of the p65 Subunit to Transactivate Gene Expression
J. Biol. Chem.,
February 4, 2000;
275(5):
3114 - 3120.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. P. Lian, P. G. Marks, J. Y. Wang, D. L. Falls, and J. A. Badwey
A Protein Kinase from Neutrophils That Specifically Recognizes Ser-3 in Cofilin
J. Biol. Chem.,
January 28, 2000;
275(4):
2869 - 2876.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Lewis, C. Di Ciano, O. D. Rotstein, and A. Kapus
Osmotic stress activates Rac and Cdc42 in neutrophils: role in hypertonicity-induced actin polymerization
Am J Physiol Cell Physiol,
February 1, 2002;
282(2):
C271 - C279.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
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ABSTRACT
INTRODUCTION