 |
INTRODUCTION |
The small GTPases Rac1 and Rac2 are members of the Rho subfamily
of the Ras-related GTP-binding proteins and serve as a molecular switch
cycling between an active GTP-bound and an inactive GDP-bound state
(1-3). In the active state, Rac interacts with a variety of effector
proteins to elicit cellular responses, including cytoskeletal reorganization and gene activation (1-3). Growth factor or
integrin-induced Rac activation in such responses is thought to require
PtdIns(3,4,5)P3,1
that is generated from PtdIns(4,5)P2 by PI3K (4), based on experiments using cells overexpressing constitutively active and dominant negative forms of Rac, and on those of microinjection of
the mutant Rac proteins to cells (1-4). A major problem in this field
has been the lack of the assay to evaluate activation of endogenous Rac.
In human neutrophils, Rac is considered to be involved in activation of
the phagocyte NADPH oxidase. The oxidase, dormant in resting cells, is
activated during phagocytosis of invading microorganisms to produce
superoxide, a precursor of microbicidal oxidants, thereby playing an
important role in host defense (5-7). The activation, at a cell level,
can be mimicked by soluble stimuli (5-8) including the chemotactic
peptide fMLP and LTB4, which bind to their own
Gi-coupled receptors on the plasma membrane (8-10), and
PMA, an activator of PKC (11). The NADPH oxidase is also activated with
anionic amphiphiles such as arachidonic acid in a cell-free system
reconstituted with five polypeptides; the membrane-bound catalytic core
cytochrome b558 comprising the two subunits
gp91phox and p22phox, and
the three cytosolic signaling proteins p47phox,
p67phox, and Rac (5-7). In the system, solely
the GTP-bound Rac, but not the GDP-bound protein, is able to induce
superoxide production (12-14), probably via binding to
p67phox (15-17). This implicates that Rac
functions as a switch for the oxidase activation, although the oxidase
activation also requires Rac-independent events such as
stimulus-induced conformational change of
p47phox that leads to its interaction with
p22phox (18-21). In Epstein-Barr
virus-transformed B lymphocytes or HL60 leukemic cells, introduction of
Rac antisense oligonucleotides or expression of a dominant negative
form of Rac2 is shown to partially inhibit superoxide production (22,
23), suggesting a role of Rac on the oxidase activation at a cell
level. However, stimulus-dependent activation of Rac has
not been demonstrated.
Activation-specific probes for small GTPases have recently been
constructed, which allows determination of the activity of endogenous
Ras and Rap1 (24-26) and hemagglutinin-tagged Rac1 and Cdc42 (27)
without radioactive in vivo labeling. Using a similar procedure, we have developed here a novel assay to estimate activation of endogenous Rac in cells, by pulling down the GTP-bound, active Rac
with the Rac-binding domain (RBD) of the protein kinase PAK2 expressed
as a GST fusion. This assay is based on the finding that PAK2-RBD binds
to the GTP-bound Rac with a high affinity, whereas the affinity for the
GDP-bound protein is undetectably low (28). Using this method, we show
here that Rac2, the predominant isoform in human neutrophils (29), is
rapidly and transiently converted to the GTP-bound, active state, in
response to the Gi-coupled receptor agonists fMLP and
LTB4. The activation appears to require PI3K that is known
to be stimulated by the Gi signaling in neutrophils (8,
30-33). On the other hand, PMA induces a slow but more sustained activation of Rac2, which is dependent on PKC but independent of
PI3K.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
fMLP and wortmannin were purchased from Wako
Chemical (Osaka, Japan), LTB4 from Cayman Chemical (Ann
Arbor, MI), PMA and pertussis toxin (PTX) from Research Biochemicals
International (Natick, MA), and LY294002 and GF109203X from Biomol
Research Laboratories (Plymouth Meeting, PA). Other chemicals used were
of the highest purity commercially available.
Preparation of the Rac-binding Domain of Human PAK2 as a GST
Fusion Protein--
We isolated a DNA fragment encoding the
Rac-binding domain of human PAK2 (PAK2-RBD; amino acids 66-147) from
total RNA of the neuroblastoma cell line SH-SY5Y by reverse
transcriptase-polymerase chain reaction, according to the protocol of
the manufacturer (Perkin Elmer). The polymerase chain reaction product
was subcloned into pGEX-4T (Amersham Pharmacia Biotech), and subjected
to DNA sequencing for the confirmation of precise construction. The GST fusion protein was expressed in E. coli BL21 cells and
purified by glutathione-Sepharose-4B (18, 20, 21).
Preparation of Recombinant Human Small GTPases--
The DNA
fragments encoding human Cdc42 and RhoA were obtained by reverse
transcriptase-polymerase chain reaction and cloned into pGEX-2T
(Amersham Pharmacia Biotech). The cDNAs for human Rac1 and Rac2
subcloned into pGEX-2T were prepared as described previously (17). The
identities of all the constructs were verified by DNA sequencing. The
GST fusion proteins were prepared as described above, followed by
cleavage with thrombin, according to the protocol of the manufacturer
(Amersham Pharmacia Biotech).
Specific Detection of the GTP-bound Rac by
GST-PAK2-RBD--
Recombinant Rac1 or Rac2 was incubated for 30 min at
30 °C in 90 µl of a nucleotide-exchange buffer (137 mM
NaCl, 2.7 mM KCl, 2 mM EDTA, 4.3 mM
Na2HPO4, and 1.4 mM
KH2PO4, pH 7.0) in the presence of 11 mM GTP
S, GTP, GDP, ATP, CTP, or UTP. The exchange
reaction was terminated by the addition of 10 µl of 100 mM MgCl2. The nucleotide-loaded proteins were
incubated at 4 °C with GST-PAK2-RBD and glutathione-Sepharose-4B beads in buffer A (20 mM Hepes, pH 7.4, 142.5 mM NaCl, 1% Nonidet P-40, 10% glycerol, 4 mM
EGTA, 4 mM EDTA). After washing the beads three times with
buffer A, the samples were subjected to 12% SDS-PAGE, and stained with
Coomassie Brilliant Blue.
Assay for Rac2 Activation in Human Neutrophils--
Human
neutrophils were isolated from fresh venous blood of healthy volunteers
by dextran sedimentation, hypotonic lysis, and the Conray/Ficoll method
(10, 18). More than 98% of the cells were neutrophils in the preparation.
The neutrophils (2.8 × 107 cells) in 700 µl of
Hepes-buffered saline (HBS; 20 mM Hepes, pH 7.4, 135 mM NaCl, 5 mM KCl, 2 mM glucose, 1 mM MgSO4, and 0.6 mM
CaCl2) were preincubated in the presence (for stimulation
with fMLP or LTB4) or absence (for stimulation with PMA) of
cytochalasin B (5 µg/ml) at 37 °C for 5 min and subsequently stimulated with indicated concentrations of fMLP, LTB4, or
PMA. Where indicated, human neutrophils were preincubated for 2 h
at 37 °C in the presence or absence of PTX (8 µg/ml), followed by stimulation with fMLP, LTB4, or PMA. The reaction was
stopped by the addition of the same volume of the lysis buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 2% Nonidet
P-40, 20% glycerol, 8 mM EGTA, 8 mM EDTA, 80 µM p-amidinophenylmethanesulfonyl fluoride (hydrochloride), 100 µg/ml of aprotinin, and 200 µg/ml each of leupeptin, chymostatin, and pepstatin A).
The lysate was centrifuged for 20 s at 12,000 × g, and the supernatant was incubated on ice for 3 min with
GST-PAK2-RBD, which had been freshly coupled with
glutathione-Sepharose-4B beads. Proteins complexed with the beads were
recovered by centrifugation, washed two times with buffer A, and
resuspended in Laemmli sample buffer. The proteins were resolved by
12% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane
(Millipore). The membrane was probed with anti-Rac1 antibody (C-11) or
anti-Rac2 antibody (C-11) (Santa Cruz Biotechnology), and detection was
performed using horseradish peroxidase-conjugated donkey anti-rabbit
antibody and the ECL plus detection kit (both from Amersham Pharmacia Biotech).
Assay for Superoxide Production by Neutrophils--
Human
neutrophils suspended in 1 ml of HBS were preincubated for 5 min at
37 °C and subsequently stimulated with PMA or fMLP. The
superoxide-producing activity was determined, as described previously
(10, 18).
 |
RESULTS |
The Rac GTPases in the GTP-bound State, but Not in the GDP-bound
State, Bind to GST-PAK2-RBD--
To test the validity of the RBD of
human PAK2 (amino acids 66 to 147) as a tool to identify the active
GTP-bound state of Rac, we expressed and purified the domain as a GST
fusion protein (GST-PAK2-RBD). Recombinant Rac1 and Rac2 preloaded with
GTP
S were precipitated using glutathione-Sepharose-4B beads coupled to the fusion protein, washed three times, and analyzed by SDS-PAGE (Fig. 1). Under the conditions, the
binding of the active Rac1/2 to GST-PAK2-RBD appeared stable because
further washing did not affect the recovery of the GTPases (data not
shown). The same results were obtained when GTP was loaded instead of
GTP
S (data not shown). On the other hand, the beads coupled to
GST-PAK2-RBD did not retain the wild-type Rac1 and Rac2 in GDP-bound
states (Fig. 1). When Rac2 was loaded with ATP, CTP, or UTP, no
interaction with the GST fusion protein was observed (data not shown).
In addition, neither GTP- nor GTP
S-bound Rac interacted with the beads coupled to GST alone (data not shown). Thus PAK2-RBD specifically recognizes the GTP-bound, active state of Rac to form a stable complex
and thereby can be used as an activation-specific probe for Rac.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 1.
Binding of GST-PAK2-RBD to the GTP-bound form
of the Rac GTPase in vitro. Recombinant Rac1 or
Rac2, preloaded with GTP S or GDP, was incubated with GST-PAK2-RBD
and glutathione-Sepharose-4B beads. After washing the beads, the
proteins were subjected to 12% SDS-PAGE and stained with Coomassie
Brilliant Blue.
|
|
Rac2 Is Activated in Human Neutrophils Stimulated with fMLP,
LTB4, and PMA--
After human neutrophils were stimulated
with the Gi-coupled receptor agonist fMLP and then lysed,
Rac was precipitated with GST-PAK2-RBD bound to glutathione-Sepharose
beads and identified by Western blot with a polyclonal antibody against
Rac2. This antibody is specific to Rac2 and does not recognize other
members of the Rho family GTPases such as Rac1, Cdc42, or RhoA (data
not shown). As shown in Fig.
2A, stimulation with fMLP led
to a rapid and transient increase in the amount of Rac2 that bound to
PAK2-RBD: the increase occurred within 30 s and reached its
maximum level at 1 min. The amounts of Rac2 detected were dependent on
the concentration of fMLP (Fig. 2B). Increases do not seem
to be because of changes in Rac protein levels because the same amount
of Rac2 was detected during the stimulation in the whole cell lysates
by Western blot analysis (data not shown). At the maximal level, about
10-12% of total Rac2 could be precipitated with GST-PAK2-RBD, when
estimated by Western blot using various amounts of the whole cell
lysates (Fig. 2C). In contrast with the GST fusion protein,
GST alone bound to the beads failed to precipitate Rac2 in the
fMLP-stimulated cells (data not shown). When an antibody specific to
Rac1 was used instead of the anti-Rac2 antibody, no specific bands on
Western blot were observed (data not shown). This is consistent with
Rac2 being largely predominant (>96%) in human neutrophils (29). Since PAK2-RBD associates exclusively with the GTP-bound state of Rac2,
with no detectable affinity for the GDP-bound protein (Fig. 1), we
conclude that fMLP induces a rapid and transient conversion of Rac2 to
the GTP-bound active state. LTB4, another Gi-coupled receptor agonist, also caused a rapid and
transient activation of Rac2 in a dose-dependent manner
(Fig. 2, A and B). The maximal amount of the
GTP-bound Rac2 formed by LTB4 (about 10% of total Rac2)
was slightly less than that in response to fMLP (Fig.
2C).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 2.
Rac2 activation in human neutrophils
stimulated with fMLP, LTB4, or PMA.
A, human neutrophils were stimulated for the indicated time
with fMLP (1 µM), LTB4 (1 µM),
or PMA (1 µg/ml). B, neutrophils were stimulated for 1 min
with fMLP or LTB4, or for 10 min with PMA at indicated
concentrations. After the incubation, the cells were lysed, and the
GTP-bound, active Rac2 was precipitated with GST-PAK2-RBD bound to
glutathione-Sepharose-4B beads. Rac2 was identified by Western blot
with an anti-Rac2 antibody. For details, see "Experimental
Procedures." C, neutrophils were stimulated for 1 min with
fMLP (1 µM) or LTB4 (1 µM), or
for 10 min with PMA (1 µg/ml). After the incubation, the cells
(2.8 × 107 cells) were lysed, and the GTP-bound,
active Rac2 was precipitated with GST-PAK2-RBD bound to
glutathione-Sepharose-4B beads. The precipitates (2.8 × 107 cell equivalents) or the whole cell lysates (derived
from the indicated cell numbers of human neutrophils) were subjected to
12% SDS-PAGE and analyzed by Western blot.
|
|
Human neutrophils, in response to fMLP, produce superoxide, the
production which is catalyzed by the phagocyte NADPH oxidase that is
activated upon cell stimulation (31-34). LTB4 also
triggers superoxide production but to a lesser extent (10). The
kinetics of the production by these agonists, i.e. rapid
onset and short duration of 1-2 min (10, 32, 34), bear a resemblance
to those of Rac2 activation (Fig. 2A), which suggests that
the agonist-induced conversion of Rac2 to the active state is linked to
the NADPH oxidase activation. We next tested whether PMA, a potent
inducer of superoxide production (34), affects states of Rac2 in
neutrophils or not. As shown in Fig. 2B, PMA induced Rac2
activation in a dose-dependent manner. The activation
occurred slowly but in a more sustained manner: it was observed as
early as 1-2 min and reached a maximum after 5-10 min, followed by a
decrease at 20 min (Fig. 2A). The time course also resembles
that of PMA-triggered activation of the NADPH oxidase (Ref. 34; and
data not shown). Approximately 7-8% of total Rac2 was converted to
the GTP-bound state by PMA at the maximum (Fig. 2C).
Effects of Pertussis Toxin on Rac2 Activation in Human
Neutrophils--
It is well documented that both fMLP and
LTB4 receptors on human neutrophils are coupled to the
Gi class of heterotrimeric G-proteins (8, 9). The finding
that both agonists cause activation of Rac2 in neutrophils (Fig. 2)
suggests a role for Gi. To clarify the involvement of
Gi in Rac2 activation, we tested the effect of PTX, which
catalyzes ADP-ribosylation of Gi
to uncouple
Gi from the receptors (8). The toxin treatment of human
neutrophils resulted in a complete loss of the fMLP-induced superoxide
production, while it did not affect the PMA-induced one (data not
shown). In the PTX-treated cells, neither fMLP nor LTB4 was
capable of activating Rac2 (Fig. 3),
indicating that coupling of the receptors to Gi is required
for the Rac2 activation. On the other hand, the treatment did not
affect PMA-triggered activation of Rac2 (Fig. 3).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3.
Effects of pertussis toxin
on Rac2 activation in human neutrophils. Human neutrophils were
preincubated for 2 h at 37 °C with or without PTX (8 µg/ml)
and then stimulated for 1 min with fMLP (1 µM),
LTB4 (1 µM), or for 10 min with PMA (1 µg/ml). The GTP-bound, active Rac2 was detected as described in the
legend to Fig. 2.
|
|
Effects of PI3K Inhibitors on Rac2 Activation in Human
Neutrophils--
In human neutrophils, fMLP activates PI3K to generate
PtdIns(3,4,5)P3 (30-33). PI3K inhibitors block not only
the generation of PtdIns(3,4,5)P3, but also superoxide
production induced by fMLP (31-33). To investigate the role of PI3K in
Rac2 activation, we used wortmannin and LY294002, that specifically
inhibit PI3K by distinct mechanisms (35, 36). As shown in Fig.
4, A and B,
fMLP-induced Rac2 activation was dose-dependently inhibited by wortmannin and by LY294002, respectively. Both inhibitors blocked fMLP-triggered superoxide production (Refs. 31-33; and data not shown)
and PtdIns(3,4,5)P3 generation (31-33, 35) in the same dose-dependent manner. LTB4-elicited activation
of Rac2 was also sensitive to wortmannin or LY294002 (Fig.
4C). Thus the PI3K activity appears to be essential for the
Gi-mediated activation of Rac2.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of PI3K inhibitors on Rac2 activation
in human neutrophils. A and B, human
neutrophils were preincubated for 5 min with indicated concentrations
of wortmannin (wort) or LY294002 (LY) and then
stimulated for 1 min with 1 µM fMLP. C and
D, after preincubation with 1 µM wortmannin
(wort) or 100 µM LY294002 (LY),
neutrophils were stimulated for 1 min with 1 µM
LTB4 (C) or for 10 min with 1 µg/ml PMA
(D). The GTP-bound, active Rac2 was detected as described in
the legend to Fig. 2.
|
|
PMA is incapable of activating PI3K in human neutrophils (30).
Consistent with this, the PI3K inhibitors failed to affect PMA-induced
Rac2 activation (Fig. 4D) as well as superoxide production (data not shown). We therefore conclude that PMA activates Rac2 independently of PI3K.
Effect of GF109203X, a PKC Inhibitor, on Rac2 Activation--
To
study the role of PKC in PMA-induced Rac 2 activation, we tested the
effect of the bisindolylmaleimide derivative GF109203X, a potent and
selective inhibitor of PKC (37). In the presence of 0.1 µM GF109203X, the PMA-elicited superoxide production by neutrophils was reduced by about 50%, whereas fMLP was capable of
fully triggering the production (Ref. 38; and data not shown). At a
higher concentration (1.0 µM), where the PMA-elicited
superoxide production was completely prevented, the fMLP-induced one
was only partially (by about 30%) blocked (Ref. 38; and data not shown). As shown in Fig. 5, GF109203X
effectively inhibited the increase in the amount of GTP-bound Rac2 in
neutrophils stimulated by PMA but not by fMLP. Thus PMA appears to form
the GTP-bound Rac2 by activating PKC, which is not likely involved in
fMLP-triggered activation of the GTPase.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of GF109203X, an inhibitor of PKC, on
Rac2 activation in human neutrophils. Human neutrophils were
preincubated for 5 min in the absence or presence (0.1 or 1.0 µM) of GF109203X (GF) and then stimulated for 1 min with
1 µM fMLP (A) or for 10 min with 1 µg/ml PMA
(B). The GTP-bound, active Rac2 was detected as described in
the legend to Fig. 2.
|
|
 |
DISCUSSION |
In the present study, we have developed a novel method to directly
detect the GTP-bound active Rac that is converted from the GDP-bound
state in intact cells. Using this method, we show that the
Gi-coupled receptor agonists fMLP and LTB4
elicit a rapid and transient activation of Rac2 in human neutrophils
and that the activation is mediated via not only Gi but
also PI3K. On the other hand, PMA causes a slow but more sustained
activation of Rac2 in a PI3K-independent manner.
Recent studies have suggested that PI3K is located upstream of Rac in
growth factor or integrin-induced cytoskeletal reorganization (1-4).
This suggestion is, however, largely based on experiments using a
constitutively active or a dominant negative form of Rac, but not on
those evaluating extent of activation of Rac. The only exception has
been a study by Hawkins et al. (39), showing that, in
[32P]Pi-preloaded endothelial cells that
stably overexpress an epitope-tagged Rac1, platelet-derived growth
factor stimulates an increase in the [32P]GTP content of
this Rac protein to a significant but small extent (about 1.7-fold),
which is inhibited by wortmannin. The present study, using the novel
method, demonstrates that fMLP and LTB4 cause a drastic
increase in the GTP-bound, active state of "endogenous" Rac2, and
that this increase is effectively blocked by PI3K inhibitors. The
blockade does not seem to be due to nonspecific effects, because the
inhibitors do not affect PMA-induced activation of Rac2. Thus PI3K
likely functions upstream of Rac2 in the Gi-coupled
receptor signaling pathway in human neutrophils.
The mechanism by which PI3K activates Rac2 in neutrophils is presently
unknown. The Rac GTPases are directly activated by a family of GEFs
related to the oncogene product Dbl that enhance the exchange of bound
GDP for GTP on Rac (40). All known Dbl-related molecules have a PH
domain (40), and some PH domains directly interact with
PtdIns(4,5)P2 and PtdIns(3,4,5)P3, a substrate
and a product of PI3K, respectively (41). Recent studies have shown that Rac activation by GEFs, such as Vav and Sos, appears to be regulated by binding of the lipids to the PH domain (42, 43). A similar
regulation may occur in the PI3K-mediated activation of Rac2 in human
neutrophils. In addition to the PI3K-dependent mechanism,
GEFs for Rac can be regulated by posttranslational modifications such
as phosphorylation by a protein kinase. It has been reported that
tyrosine-phosphorylated Vav, but not the unphosphorylated protein,
enhances GDP/GTP exchange on Rac (44). In this context, it should be
noted that PMA, a direct activator of PKC, increases the GTP-bound,
active state of Rac2, independently of PI3K. Phosphorylation by PKC
might regulate GEF(s) to activate Rac2 in neutrophils.
Activation of Rac in stimulated neutrophils has been postulated to
occur because the GTP-bound Rac, but not the GDP-bound protein, can
activate the phagocyte NADPH oxidase in vitro (12-14). However, stimulus-dependent activation of Rac has not been
demonstrated. This study, using the novel method, shows that three
stimulants for the NADPH oxidase activation in vivo, fMLP,
LTB4, and PMA, are all capable of activating Rac2 in human
neutrophils. The kinetics of Rac2 activation by the stimulants
correspond well with those of superoxide production. Although it is
well documented that PI3K inhibitors block superoxide production
triggered by fMLP but not that by PMA (31-33), it has remained elusive
how PI3K functions in the oxidase activation. As described above, PI3K
is required for activation of Rac2 in the fMLP signaling, whereas PMA
activates Rac2 in a PI3K-independent manner. Thus the present findings
support the idea that Rac2 serves as a switch for the NADPH oxidase activation.