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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 2133-2140
Activation of the Small GTPase Rap1 in Human Neutrophils
By
Laura M'Rabet,
Paul Coffer,
Fried Zwartkruis,
Barbara Franke,
Anthony W. Segal,
Leo Koenderman, and
Johannes L. Bos
From the Laboratory for Physiological Chemistry and Department of
Pulmonary Diseases, Utrecht University, Utrecht, The Netherlands; and
the University of London Hospital, London, UK.
 |
ABSTRACT |
The small GTPase Rap1 is highly expressed in human neutrophils, but
its function is largely unknown. Using the Rap1-binding domain of
RalGDS (RalGDS-RBD) as an activation-specific probe for Rap1, we have
investigated the regulation of Rap1 activity in primary human
neutrophils. We found that a variety of stimuli involved in neutrophil
activation, including fMet-Leu-Phe (fMLP), platelet-activating factor
(PAF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and
IgG-coated particles, induce a rapid and transient Rap1 activation. In
addition, we found that Rap1 is normally activated in neutrophils from
chronic granulomatous disease patients that lack cytochrome
b558 or p47phox and have a defective NADPH oxidase system.
From these results we conclude that in neutrophils Rap1 is activated
independently of respiratory burst induction. Finally, we found that
Rap1 is activated by both the Ca2+ ionophore ionomycin
and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA),
indicating that phospholipase C (PLC) activation leading to elevated
levels of intracellular free Ca2+ and diacylglycerol
(DAG) can mediate Rap1 activation. However, inhibition of PLC and
Ca2+ depletion only marginally affected fMLP-induced Rap1
activation, suggesting that additional pathways may control Rap1
activation.
© 1998 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.
Deregulated activation of neutrophils is implicated in the pathogenesis
of a variety of inflammatory diseases leading to tissue damage.
Therefore, neutrophil function is under tight control.1-5 A
diverse array of receptors are expressed on the surface of neutrophils,
allowing regulation by a wide range of agonists. Tyrosine kinase-linked receptors (eg, granulocyte-macrophage colony-stimulating factor [GM-CSF] receptors), serpentine receptors (eg,
N-formylmethionyl-leucyl-phenylalanine [fMLP] and platelet-activating
factor [PAF] receptors), and receptors that are stimulated by immune
complexes such as the Fc receptors, activate distinct and overlapping
signaling pathways regulating neutrophil responses6,7 (and
references therein).
Several small GTPases have been implicated in controlling neutrophil
function. In particular, Rac1 was shown to play an important role in
the formation of the NADPH oxidase complex, thereby controlling the
respiratory burst.2 In addition, receptor-dependent
activation of Ras was recently demonstrated.6 Another small
GTPase suggested to play a role in neutrophil function is the Ras-like
small GTPase Rap1. Rap1 has an effector domain virtually identical to
Ras, and it has been shown that ectopic expression of Rap1, under
certain conditions, antagonizes Ras signaling. Of the two known
isozymes of Rap1, Rap1A and Rap1B, Rap1A is highly expressed in
neutrophils and has been found in a large complex with cytochrome
b588.8 However, the physiological relevance of
this association is still unknown, because Rap1 is not necessary to
reconstruct a functional NADPH-oxidase complex in vitro.9
However, it has been suggested Rap1 may mediate signaling events
controlling the respiratory burst.10,11
We have recently developed a novel assay to measure activation of Rap1,
ie, the accumulation of Rap1 in its GTP bound form.12 This
assay is based on the high affinity of the Rap1-binding domain of
RalGDS (RalGDS-RBD) for Rap1GTP, but not for Rap1GDP. In platelets, we
observed that Rap1 is very rapidly activated by  -thrombin and
various other platelet agonists. For this rapid activation, elevated
levels of intracellular calcium were necessary and
sufficient.12 To investigate the possible activation and
function of Rap1 in human neutrophils, we have analyzed which stimuli
might lead to an increase in GTP-bound Rap1. We found that Rap1 is
activated by a variety of stimuli, each with distinct kinetics,
including fMLP, PAF, GM-CSF, the phorbol ester
12-O-tetradecanoylphorbol 13-acetate (TPA), and IgG-coated particles.
Furthermore, we show that multiple signaling pathways direct the
activation of Rap1. Finally, this activation is independent of both a
functional NADPH oxidase complex and the presence of cytochrome
b588.
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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) tri-sodium citrate (pH 7.4)
-treated blood as previously described.13 Mononuclear cells
were removed by centrifugation over isotonic Percoll (1.078 g/mL) from
Pharmacia (Uppsala, Sweden). After lysis of the erythrocytes in
isotonic NH4Cl solution, neutrophils were washed and
resuspended in incubation 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) containing 0.5% human serum albumin (HSA; Central
Laboratory of The Netherlands Red Cross Blood Transfusion Service,
Amsterdam, The Netherlands). Neutrophils were incubated for 30 minutes
at 37°C before stimulation. Neutrophils isolated in this manner
were in the resting state. Neutrophils for the experiments described in
Fig 4 were isolated as described.14 In all experiments, a concentration of 107 cells/mL was used for stimulation.
Neutrophil stimulation.
One milliliter of neutrophil suspension was stimulated with one of the
following stimuli: fMLP (1 µmol/L), PAF (1 µmol/L), TPA (100 ng/mL), thapsigargin (100 nmol/L) (all from Sigma, St Louis, MO),
GM-CSF (0.1 nmol/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.15-17 In some
experiments, cells were preincubated as described in the legends of the
figures with one of the following inhibitors: IBMX, prostaglandin
E2 (PGE2), wortmannin,
staurosporine (all from Sigma), GF109203X, U73122, or LY294002 (all
from Biomol, Plymouth, PA). At different time points, 0.5 mL 3×
RIPA (1× RIPA: 150 mmol/L NaCl, 10 mmol/L Tris-HCl [pH 7.4],
1% NP-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate
[SDS], 2 mmol/L phenylmethyl sulfonyl fluoride [PMSF], 2 mmol/L
benzamidine, 4 µmol/L aprotinin, 4 µmol/L leupeptin, and 4 µg/mL
trypsin inhibitor) was added.
For stimulation with IgG-coated particles,14 500 µL of
0.8-µm latex beads (Difco, Augsburg, Germany) was washed
with phosphate-buffered saline (PBS) and suspended in PBS (pH 8.5). One
hundred microliters of human IgG (150 mg/mL; Lister Institute,
Hertfordshire, UK) was added and incubated for 30 minutes at 37°C.
Beads were washed two times with incubation buffer, resuspended in 500 µL incubation buffer, and added to 108 cells in 500 µL
incubation buffer. Samples were stirred continuously and 200-µL
aliquots were lysed at indicated time points by adding 200 µL
2× RIPA.
Rap1 activation assay.
Rap1 activation was determined essentially as described.12
Cell lysates were put on ice for 8 minutes and clarified by
centrifugation at 14,000 rpm in an Eppendorf centrifuge for 8 minutes
at 4°C. Per sample, 14 µg His-Ral GDS RBD or glutathione
S-transferase (GST)-RalGDS-RBD was precoupled for 1 hour to 15 µL of
50% Ni-NTA (nickel-nitrilotriacetic acid agarose; Qiagen, Hilden,
Germany) (His-RalGDS-RBD) or 40 µL of 10% glutathione beads
(GST-RalGDS-RBD). After coupling, beads were washed 4 times with RIPA,
added to the cell lysate, and incubated for 30 minutes at 4°C.
Samples were washed 3 times in RIPA and bound proteins were eluted in 15 µL of Laemmli sample buffer. The samples were put on
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to
polyvinylidene difluoride membranes (PVDF; NEN, Boston, MA). Rap1 was
detected with a monoclonal antibody (Transduction Laboratories,
Lexington, KY) and horseradish peroxidase-coupled goat antimouse
(Bio-Rad, Hercules, CA) using enhanced chemiluminescence (Amersham,
Buckinghamshire, UK).
Depletion of intracellular free Ca2+.
Intracellular free Ca2+ was depleted as
described.18 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 (107 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 Rap1 activation and
Ca2+ measurements were performed with the same batch of
cells. Calcium concentration was measured by a dual excitation at a
wavelength of 340 nm and detected at 390 nm using Hitachi F4500
fluorescence spectrophotometer (Hitachi Ltd, Tokyo,
Japan).
Respiratory burst measurements.
Neutrophils were preincubated in the absence or presence of GM-CSF (0.1 nmol/L for 20 minutes). fMLP (1 µmol/L) was added to the cells and
oxygen consumption was measured using a Clark oxygen
electrode.19
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RESULTS |
Rap1 is activated by a variety of stimuli.
Stimulation of human neutrophils with 1 nmol/L fMLP stimulation results
in cell adhesion and chemotaxis. Antipathogenic responses such as
degranulation and the generation of oxygen radicals occur only after
stimulation with more than 1 µmol/L fMLP and require preactivation
with agents such as PAF or GM-CSF. The biochemical mechanism of this
preactivation or priming is not fully understood. Furthermore, the
priming agents PAF and GM-CSF differ in that GM-CSF stimulation results
only in chemokinesis (undirectional cell motility), whereas PAF
stimulation, similarly to fMLP stimulation results in chemotaxis
(directional cell movement)1,2,6,15-17,20 (and
references therein).
To investigate whether fMLP, PAF, or GM-CSF can activate Rap1, we
stimulated resting neutrophils isolated from human peripheral blood
with different ligands for various periods of time. Rap1 was isolated
with the 97 amino acid RalGDS-RBD, which specifically binds the active,
GTP-bound form of Rap1, followed by detection with Western
blotting.12 Stimulation with 1 µmol/L fMLP-induced a
rapid increase in Rap1 activity with biphasic kinetics
(Fig 1). An initial activation peak was
observed by 10 seconds, which decreased around 30 seconds. Activity
peaked again at 5 minutes, followed by a slow decline toward basal
levels observed in resting neutrophils. The extent of activation varied
somewhat between different donors (compare Figs 1, 3, and 4B for
variation in Rap1 activation after fMLP-stimulation), but the kinetics
of Rap1 activation remained essentially constant in all experiments.
Approximately 1% of Rap1 present in the total lysate of stimulated
cells was found to be bound to RalGDS-RBD (Fig 1). This was concluded
after comparing the amount of Rap1 present in total lysate and that of
Rap1 in the GTP bound state on Western blot. Stimulation with 1 µmol/L PAF also resulted in a rapid activation of Rap1, which peaked
around 10 seconds, followed by a slow decline towards basal level. In
contrast to fMLP, no second activation peak was observed.
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| Fig 1.
Rap1 activation in neutrophils. Neutrophils were
stimulated with either 1 µmol/L fMLP, 1 µmol/L PAF, or 0.1 nmol/L
GM-CSF for indicated time points. Cells were lysed and Rap1 GTP was
isolated using His-Ral-GDS RBD. Rap1 was detected by Western blot
analysis using a monoclonal antibody against Rap1. The amount of Rap1
GTP isolated after 10 seconds of fMLP stimulation and unstimulated
neutrophils was compared with the amount of total Rap1 (Rap1GDP and
GTP) present in various amounts (%) of total lysate. The experiments
were repeated at least three times with similar results.
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fMLP and PAF both act via serpentine receptors. To investigate whether
Rap1 could be activated via receptor-associated tyrosine kinases, we
stimulated neutrophils with GM-CSF (0.1 nmol/L). This stimulation
resulted in a delayed and weaker activation of Rap1, compared with
fMLP- or PAF-induced Rap1 activation, reaching its maximum around 5 to
10 minutes.
Various studies have implicated Rap1 functioning in the production of
oxygen radicals. Therefore, we analyzed Rap1 activation by stimuli
known to induce a respiratory burst in resting
neutrophils.14 Incubation with IgG-coated particles
resulted in a slow but steady increase of Rap1 activation detectable
after 30 seconds, which reached its maximal activity after 4 minutes
(Fig 2). A slow increase of Rap1 activation
was also observed after treating resting neutrophils with TPA (100 ng/mL). In this case, Rap1 activation was detectable after 1 minute and
reached a maximum after 5 minutes of TPA treatment.
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| Fig 2.
Rap1 activation by stimulators of the respiratory burst
in resting neutrophils. (A) Neutrophils were stimulated with 100 ng/mL
TPA or IgG-coated latex beads. Samples were taken at indicated time
points after stimulation. Rap1 activity was determined as described in
the legend to Fig 1. (B) Respiratory burst induced in resting
neutrophils by TPA (100 ng/mL) measured using a Clark oxygen
electrode.
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Activation of primed neutrophils by fMLP also induces the respiratory
burst (Fig 3A). To investigate whether Rap1
activation is modified under these conditions, we primed neutrophils
with 0.1 nmol/L GM-CSF followed by stimulation with 1 µmol/L fMLP. As
shown in Fig 3B, fMLP induced Rap1 activation to a similar extent both
in resting and in primed neutrophils. In general, the
kinetics of activation were slightly different, with the first activation peak delayed and the absence of a second peak in the case of
primed cells. As a consequence, usually only a single activation peak
was observed around 30 seconds, which decreased after 1 minute. Similar
results were obtained after priming neutrophils with PAF (data not
shown).
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| Fig 3.
Rap1 activation in resting and primed neutrophils. Rap1
activity is measured in primed and resting neutrophils. Neutrophils
were primed by 20 minutes of 0.1 nmol/L GM-CSF stimulation.
Subsequently, resting and primed neutrophils were stimulated with 1 µmol/L fMLP for the indicated time points. (A) Oxygen consumption was
measured with a Clark oxygen electrode. (B) Rap1 activation was
measured in neutrophils from the same donor as (A). Representative
examples of at least three independent experiments are shown.
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Multiple signaling pathways direct fMLP-induced Rap1 activation.
Although Rap1 was activated by all agents used, the kinetics of Rap1
activation differed, suggesting that multiple signaling pathways might
regulate Rap1 activation. To determine the mechanisms by which Rap1
GTPase is regulated, we investigated fMLP-induced Rap1 activation,
because some of the signaling pathways used by fMLP in resting
neutrophils are defined. fMLP activates a serpentine receptor that is
coupled to various heterotrimeric G-proteins (Bokoch7 and
references therein). After stimulation, phospholipase C  (PLC) is
activated, resulting in diacylglycerol (DAG)-mediated activation of
protein kinase C (PKC) and inositol 3 4 5 triphosphate (IP3)-mediated Ca2+
mobilization.7,21 In addition,
phosphatidylinositol-3-kinase (PI-3K) is activated, which may be
responsible for the activation of protein kinase B (PKB) and of Rac
GTPases.21-23
A possible signaling pathway which may mediate Rap1 activation involves
changes in intracellular free Ca 2+ concentration
([Ca2+]i). We first investigated whether
Ca2+ was able to induce Rap1 activation, because in human
platelets -thrombin induced Rap1 activation is
Ca2+-dependent.12 Resting neutrophils were
incubated with ionomycin and thapsigargin to mimic Ca2+
influx and Ca2+ release from internal stores. As shown in
Fig 4A, ionomycin induced a rapid
activation of Rap1 to a level similar to fMLP, whereas induction of
Rap1 by thapsigargin, although detectable, was clearly lower than that
induced by fMLP. Because these experiments indicated that
Ca2+ influx may be sufficient to induce Rap1 activation in
neutrophils, we next investigated whether an increase in
[Ca2+]i was essential for Rap1 activation. We
therefore depleted neutrophils of Ca2+ by pretreatment with
1 mmol/L EGTA, 1.5 µmol/L indo-1/AM, or 100 nmol/L thapsigargin.
Under these conditions, [Ca2+]i decreased to
less than 10 nmol/L and no increase in
[Ca2+]i levels was observed after fMLP
stimulation (Fig 4B). Ca2+ depletion did not affect the
rapid fMLP-induced Rap1 activation, but we did observe a reduction in
Rap1 activation at the second activation peak as compared with the
control fMLP treatment. Similar results were obtained after inhibition
of Ca2+ influx by blocking Ca2+ channels with
La3+ (data not shown). From these results we concluded that
elevated [Ca2+]i is sufficient but not
essential to induce Rap1 activation in human neutrophils.
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| Fig 4.
Ca2+ dependency of Rap1 activation. (A)
Neutrophils were incubated with ionomycin (100 nmol/L) or thapsigargin
(100 nmol/L) for the indicated time points. Rap1 GTP was detected as in
the legend to Fig 1. (B) Ca2+-depleted neutrophils were
stimulated with 1 µmol/L fMLP. As a control,
non-Ca2+-depleted cells were stimulated with 1 µmol/L
fMLP. Ca2+-depleted and untreated neutrophils of the same
donor were taken to measure [Ca2+]i after 1 µmol/L fMLP stimulation. Representative examples of at least three
independent experiments are shown.
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A role for PKC in Rap1 activation was suggested, because TPA induced
Rap1 activation in neutrophils (see Fig 2). Therefore, we examined
whether inhibitors of PKC could abolish fMLP-induced Rap1 activation.
However, the broad-specificity PKC inhibitors staurosporine and
GF109203X (bisindolylmaleimide) did not inhibit fMLP-induced Rap1
activation, indicating that the fMLP-induced Rap1 activation is
independent of PKC (Fig 5A). Furthermore,
most of the TPA-induced Rap1 activation was insensitive to
staurosporine, whereas, at the concentrations used,
staurosporine effectively abolished TPA-induced respiratory burst (Fig
5B). Thus, Rap1 can be activated directly by TPA and does not require
PKC.
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| Fig 5.
fMLP- and TPA-induced Rap1 activation is independent of
PKC. (A) fMLP-induced Rap1 activation is not inhibited by PKC
inhibitors. Neutrophils were preincubated for 5 minutes with 200 nmol/L
staurosporine or 10 minutes with 5 µmol/L GF109203X. Neutrophils were
then stimulated with 1 µmol/L fMLP for 10 seconds and 5 minutes. As a
control, untreated neutrophils of the same donor were used. (B)
TPA-induced Rap1 activity is not inhibited by a PKC inhibitor.
Neutrophils were stimulated with 100 ng/mL TPA for 5 and 10 minutes
after preincubation for 5 minutes with 200 nmol/L staurosporine or
buffer. Oxygen consumption was measured with a Clark oxygen electrode
to measure the functionality of staurosporine treatment. Representative
examples of at least three independent experiments are shown. (C)
Inhibition of PLC does not influence Rap1 activity. Neutrophils were
preincubated with 1 µmol/L U73122 (PLC inhibitor) for 3 minutes
and subsequently stimulated with 1 µmol/L fMLP. (D) Rap1 activity is
not inhibited by PI-3 kinase inhibitors. Neutrophils were preincubated
with the PI-3 kinase inhibitors LY294002 (10 µmol/L) or wortmannin
(100 nmol/L) for 5 minutes. Samples were taken after preincubation and
10 seconds and 5 minutes after 1 µmol/L fMLP stimulation. As a
control, neutrophils of the same donor without preincubation were used.
Representative examples of at least three independent experiments are
shown.
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Because both elevated levels of [Ca2+]i and
TPA-treatment (which mimics the formation of DAG) activated Rap1,
PLC may mediate fMLP-induced Rap1 activation. We investigated
whether the PLC-inhibitor U7312224 could inhibit
fMLP-induced Rap1. However, no inhibition of fMLP-induced Rap1
activation was observed (Fig 5C). From these results we concluded that
another, distinct signaling pathway regulating Rap1 activation is
activated by fMLP or that a U73122-insensitive PLC isoform is
responsible for the fMLP-induced Rap1 activation. As expected, both
inhibition of PKC and Ca2+ depletion did not abolish
fMLP-induced Rap1 activation either (Fig 5C).
To investigate whether Rap1 activation is mediated by
PI-3K,6 we treated resting neutrophils with the PI-3K
inhibitors wortmannin and LY294002. Both inhibitors failed to inhibit
fMLP-induced Rap1 activation under conditions that did abolish
respiratory burst (Fig 5D and data not shown).
Rap1 activation is not inhibited by PGE2 and is
independent of oxidase assembly and function.
In platelets, Rap1 activation by -thrombin is completely inhibited
by prostacylin, a platelet antagonist that elevates the levels of cAMP.
PGE2 is an antagonist of neutrophils and also elevates the
levels of cAMP. In addition, PGE2 was reported to induce
Rap1 phosphorylation via cAMP-dependent protein kinase A (PKA),
resulting in the dissociation of Rap1 from cytochrome b558
in vitro.25 We therefore examined if PGE2 could
antagonize fMLP-dependent Rap1 activation in neutrophils. However,
cotreatment of neutrophils with PGE2 and the
phosphodiesterase inhibitor IBMX (which further elevates cAMP levels)
did not affect fMLP-induced Rap1 activation
(Fig 6). The observation that a similar
treatment did inhibit fMLP-induced respiratory burst in GM-CSF-primed
neutrophils indicated that cAMP does not interfere with the activation
of Rap1 GTPase.
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| Fig 6.
Rap1 activation is not inhibited by PGE2
treatments. Neutrophils of a healthy donor were preincubated for 10 minutes with 100 µmol/L IBMX followed by 30 seconds of incubation
with 30 µmol/L PGE2. Cells were stimulated with 1 µmol/L fMLP, and Rap1 activity was measured after the indicated time
points. As a control, untreated neutrophils were stimulated
with the same amount of fMLP. Respiratory burst was measured to control
for the functionality of the cAMP treatment. Representative examples of
at least three independent experiments are shown.
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That Rap1 activation was not dependent on Rap1 association with
cytochrome b558 or the formation of the oxidase complex was further confirmed by analysis of Rap1 activation in patients with chronic granulomatous disease (CGD), which lack a functional oxidase complex.26 As shown in Fig 7,
Rap1 is still activated by both fMLP and TPA in neutrophils isolated
from CGD patients, lacking either cytochrome b588 (p91phox)
or p47phox.
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| Fig 7.
Rap1 activation is independent of the presence of a
functional NADPH oxidase complex. (A) Neutrophils of a
p91phox-deficient or a p47-deficient patient were stimulated for 5 minutes with 100 ng/mL TPA or 1 µmol/L fMLP. Rap1 GTP was isolated
using GST-Ral-GDS RBD as described in the legend to Fig 1.
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DISCUSSION |
In this report we have investigated the signaling events in human
neutrophils that resulted in the activation of Rap1, ie, an increase in
levels of Rap1GTP. We have used an assay that uses the Rap1 binding
domain of a putative Rap1 effector (RalGDS-RBD) to specifically
precipitate Rap1GTP. We observed that a variety of stimuli can induce
Rap1 activation, but the extent and kinetics of activation varied. A
very rapid activation of Rap1 was observed after fMLP and PAF
stimulation, whereas slower activation was observed after stimulation
with TPA, incubation with IgG-coated particles, or GM-CSF.
Our finding that several stimuli can activate Rap1 may indicate that
multiple pathways are involved in the activation of Rap1. Thus far, a
study of the activation of Rap1 has only been performed in human
platelets and T lymphocytes. In these cells, Rap1 activation after
-thrombin stimulation is mediated by elevated
[Ca2+]i, which is necessary and
sufficient.12,27 In neutrophils, it appears that, in
addition to elevated [Ca2+]i, other pathways
lead to Rap1 activation. Indeed, both ionomycin and, to a lesser
extent, thapsigargin induce Rap1 activation, but Ca2+
depletion did not abolish fMLP-induced Rap1 activation as well.
Because TPA efficiently induced Rap1 activation, DAG is a strong
candidate to mediate Rap1 activation. PKC, a well-known target for DAG,
is not involved in Rap1 activation, because both the fMLP-induced Rap1
activation and the TPA-induced Rap1 activation were not abolished after
treatment with the broad PKC inhibitor staurosporine. This implies that
a different DAG target is involved in the regulation of Rap1.
Suprisingly, affecting the elevation of
[Ca2+]i and DAG formation by inhibition of
PLC did not abolish fMLP-induced Rap1 activation. This could mean
that U73122 is not sufficient to block an increase in
[Ca2+]i and/or the formation of DAG.
Indeed, PLC-independent pathways for Ca2+ influx and DAG
formation have been described.7,28,29 Alternatively, a
third, still-unknown pathway is involved in the regulation of fMLP-induced Rap1 activation.
It is unlikely that fMLP-induced Rap1 activation is mediated by
PI-3K,24,30 because the PI-3K inhibitors wortmannin and LY294002 also failed to abolish Rap1 activation either.
It was shown previously that Rap1 is present in a complex with
cytochrome b558 of the NADPH oxidase and cotranslocates
with cytochrome b558 from the specific granules to the
plasma or phagosome membrane after stimulation.10,11 Our
results show that association of Rap1 with cytochrome b558
or an assembled oxidase complex is not necessary for Rap1 activation.
This conclusion was based on our observation that Rap1 is normally
activated in neutrophils from CGD patients that lack either p91phox or
p47phox. Furthermore, PGE2, which was reported to induce
Rap1 phosphorylation and dissociation from cytochrome
b558,25 did not abolish Rap1 activation.
Our finding that Rap1 is activated after stimulation of both receptor
associated-tyrosine kinases (GM-CSF) and serpentine receptors (fMLP and
PAF) indicates that Rap1 functions in a pathway common to both receptor
types. Interestingly, only GM-CSF but not fMLP induces phosphorylation
of Clb.31 This protein has been implicated in the
activation of Rap1 through the recruitment of C3G, an exchange factor
for Rap1, to Cbl via the adaptor protein Crk or Crk-L.32,33
However, if indeed Cbl mediates Rap1 activation, pathways inducing Rap1
activation after fMLP stimulation would be independent of Cbl
phosphorylation.
Although the function of Rap1 is unknown, it has been suggested that
Rap1 activity is required for respiratory burst.10,11 Our
results show that Rap1 activation in vivo is not sufficient to induce a
respiratory burst. Perhaps Rap1 activation is only involved in one of
the events regulating the multistep process of generating a respiratory
burst. Rap1 activity has also been found in human
platelets.12 Both neutrophils and platelets are highly
specialized, and Rap1 is possibly involved in a specific function
common to both cell types. The reported association of Rap1 to
cytochrome b588, in human neutrophils may indicate that Rap1 is involved in the regulation of complex formation. In platelets, a close relative of Rap1, Rap2, has been found in a complex with the
major platelet integrin
IIb3,34 suggesting a similar
role for Rap2 in platelets. Furthermore, in both cell types, Rap1 is at
least partly localized on vesicular structures and is translocated to
the plasma membrane upon activation.35 Interestingly, the Rap homologue in the budding yeast Saccharomyces
cerevisiae, Rsr1 (Bud1) is involved in the selection
of the site where a new bud will form.36 Perhaps Rap1
regulates the formation and/or translocation of protein
complexes by specifying the fusion of different cellular compartments,
such as the fusion of secretory vesicles with the plasma membrane. It
is suggested that Rap1 is involved in regulated insulin
secretion.37
To decipher the function of Rap1 it will be essential to know which
effector protein binds to Rap1GTP. Interestingly, the effector domain
of Rap1 is virtually identical to the effector domain of
Ras,38-40 and it has been shown that Rap1 also binds to Ras
effector proteins, including Ral guanine nucleotide exchange factors
(RalGEFs) and Raf kinases.41-44 In neutrophils and
platelets, Rap1 may activate one of the Raf1 kinase signaling
cascades,43,44 or Rap1 may activate one of the RalGEFs
resulting in the activation of the small GTPase Ral.41,45
This protein is involved in the activation of phospholipase
D,46 which, together with members of the Arf family of
small GTPases, may control translocation and fusion of
granules.47
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FOOTNOTES |
Submitted February 28, 1998;
accepted May 14, 1998.
Supported by grants from the Netherlands Heart Association (B.F.), the
Dutch Cancer Society (F.Z.), and Glaxo Wellcome (P.C.).
Address reprint requests to Johannes L. Bos, PhD, Laboratory for
Physiological Chemistry, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands; e-mail: j.l.bos{at}med.ruu.nl.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Drs B. Burgering and K. Reedquist for support,
discussions, and critically reading the manuscript.
| |
REFERENCES |
1.
DeLeo FR,
Quinn MT:
Assembly of the phagocyte NADPH oxidase: Molecular interaction of oxidase proteins.
J Leukoc Biol
60:677,
1996[Abstract]
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.
Haslett C,
Savill JS,
Meagher L:
The neutrophil.
Curr Opin Immunol
2:10,
1989[Medline]
[Order article via Infotrieve]
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.
Coffer PJ,
Geijsen N,
M'Rabet L,
Schweizer RC,
Maikoe T,
Raaijmakers JAM,
Lammers JWJ,
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
7.
Bokoch GM:
Chemoattractant signaling and leukocyte activation.
Blood
86:1649,
1995[Free Full Text]
8.
Quinn MT,
Parkos CA,
Walker L,
Orkin SH,
Dinauer MC,
Jesaitis AJ:
Association of a Ras-related protein with cytochrome b of human neutrophils.
Nature
342:198,
1989[Medline]
[Order article via Infotrieve]
9.
Abo A,
Boyhan A,
West I,
Thrasher AJ,
Segal AW:
Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245.
J Biol Chem
267:16767,
1992[Abstract/Free Full Text]
10.
Gabig TG,
Crean CD,
Mantel PL,
Rosli R:
Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation.
Blood
85:804,
1995[Abstract/Free Full Text]
11.
Maly FE,
Quilliam LA,
Dorseuil O,
Der CJ,
Bokoch GM:
Activated or dominant inhibitory mutants of Rap1A decrease the oxidative burst of Epstein-Barr virus-transformed human B lymphocytes.
J Biol Chem
269:18743,
1994[Abstract/Free Full Text]
12.
Franke B,
Akkerman J-WN,
Bos JL:
Rapid Ca2+-mediated activation of Rap1 in human platelets.
EMBO J
16:252,
1997[Medline]
[Order article via Infotrieve]
13.
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]
14.
Segal AW,
Coade SB:
Kinetics of oxygen consumption by phagocytosing human neutrophils.
Biochem Biophys Res Commun
84:611,
1978[Medline]
[Order article via Infotrieve]
15.
Dewald B,
Baggiolini M:
Activation of NADPH oxidase in human neutrophils. Synergism between fMLP and the neutrophil products PAF and LTB4.
Biochem Biophys Res Commun
128:297,
1985[Medline]
[Order article via Infotrieve]
16.
Ingraham LM,
Coates TD,
Allen JM,
Higgins CP,
Baehner RL,
Boxer LA:
Metabolic, membrane, and functional responses of human polymorphonuclear leukocytes to platelet-activating factor.
Blood
59:1259,
1982[Abstract/Free Full Text]
17.
Weisbart RH,
Kwan L,
Golde DW,
Gasson JC:
Human GM-CSF primes neutrophils for enhanced oxidative metabolism in response to the major physiological chemoattractants.
Blood
69:18,
1987[Abstract/Free Full Text]
18.
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]
19.
Weening RS,
Roos D,
Loos JA:
Oxygen consumption of phagocytizing cells in human leukocyte and granulocyte preparations: A comparative study.
J Lab Clin Med
83:570,
1974[Medline]
[Order article via Infotrieve]
20.
Edwards SW:
Cell signalling by integrins and immunoglobulin receptors in primed neutrophils.
Trends Biochem Sci
20:362,
1995[Medline]
[Order article via Infotrieve]
21.
Thelen M,
Wirthmueller U:
Phospholipases and protein kinases during phagocyte activation.
Curr Opin Immunol
6:106,
1994[Medline]
[Order article via Infotrieve]
22.
Didichenko SA,
Tilton B,
Hemmings BA,
Ballmer-Hofer K,
Thelen M:
Constitutive activation of protein kinase B and phosphorylation of p47phox by a membrane-targeted phosphoinositide 3-kinase.
Curr Biol
6:1271,
1996[Medline]
[Order article via Infotrieve]
23.
Tilton B,
Andjelkovic M,
Didichenko SA,
Hemmings BA,
Thelen M:
G-protein-coupled receptors and Fcgamma-receptors mediate activation of Akt/protein kinase B in human phagocytes.
J Biol Chem
272:28096,
1997[Abstract/Free Full Text]
24.
Ding J,
Vlahos CJ,
Liu R,
Brown RF,
Badwey JA:
Antagonists of phosphatidylinositol 3-kinase block activation of several novel protein kinases in neutrophils.
J Biol Chem
270:11684,
1995[Abstract/Free Full Text]
25.
Bokoch GM,
Quilliam LA,
Bohl BP,
Jesaitis AJ,
Quinn MT:
Inhibition of Rap1A binding to cytochrome b558 of NADPH oxidase by phosphorylation of Rap1A.
Science
254:1794,
1991[Abstract/Free Full Text]
26.
Segal AW:
The NADPH oxidase and chronic granulomatous disease.
Mol Med Today
2:129,
1996[Medline]
[Order article via Infotrieve]
27.
Reedquist KA,
Bos JL:
Costimulation through CD28 suppresses T cell receptor-dependent activation of the Ras-like small GTPase Rap1 in human T lymphocytes.
J Biol Chem
273:4944,
1998[Abstract/Free Full Text]
28.
Thelen M,
Dewald B,
Baggiolini M:
Neutrophil signal transduction and activation of the respiratory burst.
Physiol Rev
73:797,
1993[Free Full Text]
29.
Tou J,
Jeter JR Jr,
Dola CP,
Venkatesh S:
Accumulation of phosphatidic acid mass and increased de novo synthesis of glycerolipids in platelet-activating-factor-activated human neutrophils.
Biochem J
280:625,
1991
30.
Thelen M,
Wymann MP,
Langen H:
Wortmannin binds specifically to 1-phosphatidylinositol 3-kinase while inhibiting guanine nucleotide-binding protein-coupled receptor signaling in neutrophil leukocytes.
Proc Natl Acad Sci USA
91:4960,
1994[Abstract/Free Full Text]
31.
Naccache PH,
Gilbert C,
Barabe F,
Al-Shami A,
Mahana W,
Bourgoin SG:
Agonist-specific tyrosine phosphorylation of Cbl in human neutrophils.
J Leukoc Biol
62:901,
1997[Abstract]
32.
Reedquist KA,
Fukazawa T,
Panchamoorthy G,
Langdon WY,
Shoelson SE,
Druker BJ,
Band H:
Stimulation through the T cell receptor induces Cbl association with Crk proteins and the guanine nucleotide exchange protein C3G.
J Biol Chem
271:8435,
1996[Abstract/Free Full Text]
33.
Smit L,
van der Horst G,
Borst J:
Sos, Vav, and C3G participate in B cell receptor-induced signaling pathways and differentially associate with Shc-Grb2, Crk, and Crk-L adaptors.
J Biol Chem
271:8564,
1996[Abstract/Free Full Text]
34.
Torti M,
Ramaschi G,
Sinigaglia F,
Lapetina EG,
Balduini C:
Glycoprotein IIb-IIIa and the translocation of Rap2B to the platelet cytoskeleton.
Proc Natl Acad Sci USA
91:4239,
1994[Abstract/Free Full Text]
35.
Berger G,
Quarck R,
Tenza D,
Levy-Toledano S,
de Gunzburg J,
Cramer EM:
Ultrastructural localization of the small GTP-binding protein Rap1 in human platelets and megakaryocytes.
Br J Haematol
88:372,
1994[Medline]
[Order article via Infotrieve]
36.
Chant J,
Herskowitz I:
Genetic control of bud site selection in yeast by a set of gene products that constitute a morphogenetic pathway.
Cell
65:1203,
1991[Medline]
[Order article via Infotrieve]
37.
Leiser M,
Efrat S,
Fleischer N:
Evidence that Rap1 carboxylmethylation is involved in regulated insulin secretion.
Endocrinology
136:2521,
1995[Abstract]
38.
Kawata M,
Matsui Y,
Kondo J,
Hishida T,
Teranishi Y,
Takai Y:
A novel small molecular weight GTP-binding protein with the same putative effector domain as the Ras proteins in bovine brain membranes: Purification, determination of the primary structure and characterisation.
J Biol Chem
263:18965,
1988[Abstract/Free Full Text]
39.
Pizon V,
Chardin P,
Lerosey I,
Olofsson B,
Tavitian A:
Human cDNAs Rap1 and Rap2 homologous to the Drosophila gene Dras3 encode proteins closely related to ras in the `effector' region.
Oncogene
3:201,
1988[Medline]
[Order article via Infotrieve]
40.
Kitayama H,
Sugimoto Y,
Matsuzaki T,
Ikawa Y,
Noda M:
A ras-related gene with transformation suppressor activity.
Cell
56:77,
1989[Medline]
[Order article via Infotrieve]
41.
Herrmann C,
Horn G,
Spaargaren M,
Wittinghofer A:
Differential interaction of the ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor.
J Biol Chem
271:6794,
1996[Abstract/Free Full Text]
42.
Bos JL,
Franke B,
M'Rabet L,
Reedquist K,
Zwartkruis F:
In search of a function for the Ras-like GTPase Rap1.
FEBS Lett
410:59,
1997[Medline]
[Order article via Infotrieve]
43.
Vossler MR,
Yao H,
York RD,
Pan M-G,
Rim SM,
Stork PJS:
cAMP activates MAP kinase and Elk 1 through a B-raf- and Rap1 dependent pathway.
Cell
89:74,
1997
44.
Wolthuis RMF,
Zwartkruis F,
Moen TC,
Bos JL:
Ras-dependent activation of the small GTPase Ral.
Curr Biol
8:471,
1998[Medline]
[Order article via Infotrieve]
45.
Wolthuis RM,
Franke B,
van Triest M,
Bauer B,
Cool RH,
Camonis JH,
Akkerman JW,
Bos JL:
Activation of the small GTPase Ral in platelets.
Mol Cell Biol
18:2486,
1998[Abstract/Free Full Text]
46.
Jiang H,
Luo JQ,
Urano T,
Frankel P,
Lu Z,
Foster DA,
Feig LA:
Involvement of Ral GTPase in v-Src-induced phospholipase D activation.
Nature
378:409,
1995[Medline]
[Order article via Infotrieve]
47.
Cockcroft S,
Thomas GM,
Fensome A,
Geny B,
Cunningham E,
Gout I,
Hiles I,
Totty NF,
Truong O,
Hsuan JJ:
Phospholipase D: A downstream effector of ARF in granulocytes.
Science
263:523,
1994[Abstract/Free 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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[Full Text]
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|
|
|
|
|
|
|
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[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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Small GTP-Binding Proteins
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January 1, 2001;
81(1):
153 - 208.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Katagiri, M. Hattori, N. Minato, S.-k. Irie, K. Takatsu, and T. Kinashi
Rap1 Is a Potent Activation Signal for Leukocyte Function-Associated Antigen 1 Distinct from Protein Kinase C and Phosphatidylinositol-3-OH Kinase
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March 15, 2000;
20(6):
1956 - 1969.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
Y. Alsayed, S. Uddin, S. Ahmad, B. Majchrzak, B. J. Druker, E. N. Fish, and L. C. Platanias
IFN-{gamma} Activates the C3G/Rap1 Signaling Pathway
J. Immunol.,
February 15, 2000;
164(4):
1800 - 1806.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Franke, M. van Triest, K. M. T. de Bruijn, G. van Willigen, H. K. Nieuwenhuis, C. Negrier, J.-W. N. Akkerman, and J. L. Bos
Sequential Regulation of the Small GTPase Rap1 in Human Platelets
Mol. Cell. Biol.,
February 1, 2000;
20(3):
779 - 785.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. de Rooij, N. M. Boenink, M. van Triest, R. H. Cool, A. Wittinghofer, and J. L. Bos
PDZ-GEF1, a Guanine Nucleotide Exchange Factor Specific for Rap1 and Rap2
J. Biol. Chem.,
December 31, 1999;
274(53):
38125 - 38130.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Geijsen, S. van Delft, J. A.M. Raaijmakers, J.-W. J. Lammers, J. G. Collard, L. Koenderman, and P. J. Coffer
Regulation of p21rac Activation in Human Neutrophils
Blood,
August 1, 1999;
94(3):
1121 - 1130.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. M'Rabet, P. J. Coffer, R. M. F. Wolthuis, F. Zwartkruis, L. Koenderman, and J. L. Bos
Differential fMet-Leu-Phe- and Platelet-activating Factor-induced Signaling Toward Ral Activation in Primary Human Neutrophils
J. Biol. Chem.,
July 30, 1999;
274(31):
21847 - 21852.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. A. Miller, D. L. Barber, L. L. Bell, B. K. Beattie, M.-Y. Zhang, B. G. Neel, M. Yoakim, L. I. Rothblum, and J. Y. Cheung
Identification of the Erythropoietin Receptor Domain Required for Calcium Channel Activation
J. Biol. Chem.,
July 16, 1999;
274(29):
20465 - 20472.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Tsukamoto, M. Hattori, H. Yang, J. L. Bos, and N. Minato
Rap1 GTPase-activating Protein SPA-1 Negatively Regulates Cell Adhesion
J. Biol. Chem.,
June 25, 1999;
274(26):
18463 - 18469.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. de Rooij, H. Rehmann, M. van Triest, R. H. Cool, A. Wittinghofer, and J. L. Bos
Mechanism of Regulation of the Epac Family of cAMP-dependent RapGEFs
J. Biol. Chem.,
June 30, 2000;
275(27):
20829 - 20836.
[Abstract]
[Full Text]
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A. Arai, Y. Nosaka, E. Kanda, K. Yamamoto, N. Miyasaka, and O. Miura
Rap1 Is Activated by Erythropoietin or Interleukin-3 and Is Involved in Regulation of beta 1 Integrin-mediated Hematopoietic Cell Adhesion
J. Biol. Chem.,
March 23, 2001;
276(13):
10453 - 10462.
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H. H. Versteeg, I. Hoedemaeker, S. H. Diks, J. C. Stam, M. Spaargaren, P. M. P. van Bergen en Henegouwen, S. J. H. van Deventer, and M. P. Peppelenbosch
Factor VIIa/Tissue Factor-induced Signaling via Activation of Src-like Kinases, Phosphatidylinositol 3-Kinase, and Rac
J. Biol. Chem.,
September 8, 2000;
275(37):
28750 - 28756.
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Abstract
Introduction
Abstract